Induced and activated self-delivery cyclic dinucleoside derivative and preparation method and application thereof in anti-tumor immunotherapy

Abstract

The invention provides an induced activated self-delivery cyclic dinucleoside derivative as well as a preparation method and application thereof in anti-tumor immunotherapy. The structure of the cyclic dinucleotide derivative is shown as a formula (I). The novel self-delivery ring dinucleotide STING agonist iCDN capable of being induced and activated is provided by introducing a specific group to a phosphorothioate diester site of CDN, and on-demand activation of STING is achieved. The iCDN provided by the invention can improve cell internalization efficiency and cell permeability, and in a 4T1 tumor model with poor immunogenicity, the iCDN provided by the invention can start strong immune response under biological orthogonal induction, and effectively inhibit tumor growth.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to an induced activated self-delivery cyclic dinucleoside derivative and a preparation method thereof and use thereof in anti-tumor immunotherapy. Background Art

[0002] The stimulator of interferon genes (STING) plays a key role in DNA sensing, which is used to initiate innate immune responses and is crucial for the treatment of various cancers. Cytoplasmic DNA derived from tumors or microorganisms activates cyclic GMP-AMP synthase (cGAS) to synthesize the second messenger, a cyclic dinucleotide 2',3'-cGAMP. 2',3'-cGAMP binds to STING in the endoplasmic reticulum and is transported to the Golgi apparatus, further inducing the phosphorylation of TANK binding kinase 1 (TBK1) / interferon regulatory factor 3 (IRF3) and inhibitor of kinase (IKK) / nuclear factor-KB (NF-KB) signaling pathways, inducing the release of type I interferon (IFN-I) and other inflammatory cytokines. At present, STING agonists are mainly its natural ligand cyclic dinucleotide compounds (CDN) and their analogs.

[0003] However, due to the defects of CDN such as poor cell membrane permeability, low cellular uptake rate, easy enzymatic hydrolysis and metabolic instability, their administration is greatly limited, usually intratumoral administration. In addition, due to the indifferent expression of STING protein in normal cells and tumor cells, these CDN-based agonists induce inflammatory factors in both tumor tissues and normal tissues, thereby causing systemic inflammatory responses. There are currently two main commonly used strategies. One is to use nanoparticles, liposomes and other materials to encapsulate CDN to solve the delivery problem, but this method is limited by the long-term stability, effectiveness and safety of the material itself; the other is to modify the amino, hydroxyl or phosphate bond of CDN through chemical total synthesis, but the synthesis steps are often lengthy and the overall yield is low.

[0004] Therefore, it remains a challenge to develop CDNs and their analogs that are convenient, effective, efficiently internalized into cells, and capable of controllable activation of the STING pathway. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide an induced activated self-delivering cyclic dinucleoside derivative and its preparation method and use for anti-tumor immunotherapy. The inventors have found that by introducing a specific group at the thiophosphate diester site of CDN, a new type of inducible activated self-delivering STING agonist iCDN can be obtained. The iCDN has high cell permeability, can improve cell internalization efficiency, and significantly inhibit tumor growth. Thus, the present invention provides a cyclic dinucleotide derivative and its preparation method and use.

[0006] The above object of the present invention is achieved by providing the following technical solutions:

[0007] In a first aspect, the present invention provides a cyclic dinucleotide derivative (iCDN) or a stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt thereof, wherein the structure of the cyclic dinucleotide derivative is as shown in formula (I),

[0008]

[0009] in,

[0010] R1 and R2 are each independently a purine nucleobase;

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

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

[0013]

[0014] In the present invention, the LG group can be eliminated or leave by itself under activation conditions, such as chemical orthogonal activation, light activation or enzyme activation.

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

[0016]

[0017] Preferably, R3 and R4 are both 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 according to the first aspect of the present invention or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt, comprising the following steps:

[0021]

[0022] The compound represented by formula (II) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt is reacted with the compound Br-LG to obtain the compound represented by formula (I) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide 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 invention, the method comprises the following steps:

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

[0027] (2) dissolving the compound represented by formula (II) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt in a buffer solution to obtain a second solution;

[0028] (3) The first solution and the second solution are mixed, and stirred 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, 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 stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt in the second solution is 80-150 μM, preferably 95-105 μM.

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

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

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

[0035] In a third aspect, the present invention provides a pharmaceutical composition for anti-tumor use, comprising the cyclic dinucleotide derivative according to the first aspect of the present invention or its stereoisomers, tautomers, hydrates, solvates, nitrogen oxides or pharmaceutically acceptable salts.

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

[0037] In a fourth aspect, the present invention provides use of the cyclic dinucleotide derivative or its stereoisomers, tautomers, hydrates, solvates, nitrogen oxides or pharmaceutically acceptable salts according to the first aspect of the present invention in the preparation of anti-tumor drugs.

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

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

[0040] The present invention is achieved by using CDN, such as 2', 3'-cG S A S The introduction of the LG group at the phosphorothioate diester site of MP provides a new type of inducible activation self-delivered STING agonist, iCDN, which achieves on-demand activation of STING, thereby minimizing the systemic side effects associated with nonspecific activation of STING. Specifically, when activation conditions are not provided, iCDN containing LG cannot release CDN, thereby failing to activate the STING pathway; when activation conditions are present, iCDN containing LG deprotects and releases CDN through an orthogonal reaction, thereby activating the STING pathway.

[0041] The present invention proves the feasibility of orthogonal activation of iCDN by the change of liquid phase retention time and mass spectrometry of target product peak. In addition, the present invention proves that iCDN has improved cell internalization efficiency through experiments at the cell level. Compared with cells treated with free CDN, iCDN-treated cells show significantly increased CDN levels; in addition, iCDN has high cell permeability and can increase the relative mRNA content of IFNβ and CXCL10 in cells and the phosphorylation levels of STING, TBK1 and NF-κB in cells.

[0042] In the 4T1 tumor model with poor immunogenicity, the iCDN provided by the present invention can initiate a strong immune response under bioorthogonal induction, effectively converting "cold" tumors into "hot" tumors, significantly increasing the levels of pro-inflammatory cytokines such as CXCL10, IFNβ, TNFα, IFNγ and IL6, and significantly inhibiting tumor growth. These results indicate that the inducible activation cyclic dinucleotide derivatives provided by the present invention have great potential for development into STING agonist drugs with anti-tumor efficacy.

[0043] Currently, free CDN needs to be administered intratumorally. If it is administered intravenously, it needs to be encapsulated with materials such as nanoparticles and liposomes. However, the preparation method of iCDN provided by the present invention is simple and convenient for administration. It can be administered intravenously without encapsulation with materials such as nanoparticles and liposomes, and released under specific conditions, thereby achieving controllable activation of the STING pathway. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0045] Figure 1 Schematic diagram of the synthesis and action principle of the cyclic dinucleotide derivatives of the present invention;

[0046] Figure 2 HPLC and MS images of the target peak of the iCDN-N3 reaction solution in Example 1;

[0047] Figure 3 The iCDN-N3 in Example 1 releases 2′3′-cG after activation by tri(hydroxypropyl)phosphine (THPP). s A s HPLC and MS profiles of MP;

[0048] Figure 4 It is a graph of the internalization efficiency of each group of HEK293 cells in Example 1, wherein, compared with the 2′3′-cGsAsMP group, ****P<0.0001;

[0049] Figure 5 The changes in the relative mRNA levels of IFNβ and CXCL10 in each group of HEK293 cells detected by real-time fluorescence quantitative (RT-PCR) in Example 1, where ***P<0.001, ****P<0.0001;

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

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

[0052] Figure 8 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] Fig. 9 The figure is a graph showing the body weight of each group of 4T1 tumor-bearing mice in Example 1;

[0054] Fig.10 The blood biochemical analysis diagram of each group of 4T1 tumor-bearing mice in Example 1, ns means not significant;

[0055] Fig.11 HPLC and MS images of the target peak of the iCDN-TCO reaction solution in Example 2;

[0056] Fig.12 The iCDN-TCO in Example 2 releases 2′3′-cG after being activated by 3,6-dimethyl-1,2,4,5-tetrazine s A s HPLC and MS profiles of MP;

[0057] Fig.13 It is a graph of the internalization efficiency of each group of HEK293 cells in Example 2, wherein, compared with the 2′3′-cGsAsMP group, ***P<0.001;

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

[0059] Fig.15 HPLC and MS images of the target peak of the iCDN-NB reaction solution in Example 3;

[0060] Fig.16 The iCDN-NB in ​​Example 3 releases 2′3′-cG after being activated by 365 nm light. s A s HPLC and MS profiles of MP;

[0061] Fig.17 The changes in the relative mRNA levels of IFNβ and CXCL10 in each group of HEK293 cells detected by RT-PCR in Example 3, where ***P<0.001, ****P<0.0001;

[0062] Fig.18 HPLC and MS images of the target peak of the iCDN-QP reaction solution in Example 4;

[0063] Fig.19 The iCDN-QP in Example 4 releases 2′3′-cG after NQO1 enzyme activation s A s HPLC and MS profiles of MP;

[0064] Fig. 20 is the change of relative mRNA content of NQO1 in BMDM, CT26 and 4T1 cells detected by RT-PCR in Example 4, where ****P<0.0001;

[0065] Fig.21 The changes in the relative mRNA levels of IFNβ and CXCL10 in BMDM, CT26 and 4T1 cells detected by RT-PCR in Example 4 under the activation of endogenous NQO1 enzyme by iCDN-QP, where *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below in conjunction with specific examples. The examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0067] The 2′3′-cG 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 all provided by the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences; 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 preparation method of the phosphate buffer used in the following examples is as follows:

[0069] (1) Prepare Solution A (sodium dihydrogen phosphate aqueous solution): weigh 27.6 g of NaH2PO4·H2O and dissolve it in distilled water. Add water to dilute to 1000 mL.

[0070] (2) Prepare Solution B (sodium hydrogen phosphate aqueous solution): weigh 53.6 g Na2HPO4·7H2O and dissolve it in distilled water. Add water to dilute to 1000 mL.

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

[0072] Example 1 Preparation of iCDN-N3 activated by phosphating agents and anti-tumor immunotherapy

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

[0074] 2-Methylnicotinate (1,2.0g, 2.0eq, 13.23mmol) was dissolved in 20mL of anhydrous dichloromethane (DCM), and then trichloroisocyanuric acid (3.05g, 3.0eq, 16.54mmol) was added to react, and the obtained reaction mixture was stirred at room temperature overnight, and the obtained reaction solution was purified by silica gel column to obtain compound 2 (1.0g). Compound 2 (1.0g, 1.0eq, 5.4mmol) was dissolved in 10mL of N, N-dimethylformamide (DMF), and then sodium azide (0.52g, 1.5eq, 8.1mmol) was added, and stirred at room temperature overnight to react, and the obtained reaction mixture was washed with saturated sodium bicarbonate aqueous solution, and extracted with ethyl acetate, and then the organic layer was purified by silica gel column to obtain yellow oily compound 3 (0.95g). Compound 3 (0.95 g, 5 mmol) was dissolved in 10 mL of methanol, and then 10 mL of 10 wt % NaOH aqueous solution was added. After 1 hour, the reaction was monitored by thin layer chromatography to confirm that the raw material conversion was complete. The obtained reaction solution was neutralized to obtain compound 4, which was used for 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-butyldimethylsilyl chloride (TBSCl, 6.65 g, 44.3 mmol) were added to 50 mL of N,N-dimethylformamide and stirred at room temperature overnight to react. The obtained reaction mixture was then chromatographed on a silica gel column (eluent: petroleum ether / ethyl acetate, volume ratio of 10:1) 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 obtained reaction solution was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio of 10:1) to obtain 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 react. After the reaction was quenched, dichloromethane and saturated brine in a volume ratio of approximately 1:1 were added to the reaction solution for extraction. The combined organic layer was chromatographed on silica gel (eluent: petroleum ether / ethyl acetate, volume ratio: 3:1) to obtain a white solid 8 (0.8 g, 2.3 mmol).

[0077] The synthetic route is as follows:

[0078]

[0079] 2. Small molecule compound 8 and 2′3′-cG s A s Cross-linking of MPs

[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 was dissolved in phosphate buffer to obtain a second solution, in which 2'3'-cG s A s The concentration of MP is 100 μM. The two solutions are mixed in a volume ratio of 1:1, stirred at room temperature (30°C) for 48 hours, and the resulting solution is collected and vacuum freeze-dried to remove the organic solvent. The obtained crude reaction solution is then dissolved in sterile water, analyzed and purified using high performance liquid chromatography. The liquid phase purification method is as follows:

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

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

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

[0085] 10-25 minutes: 25-45% volume fraction of phase B, the rest is phase A;

[0086] 25-80 minutes: 45-95% volume fraction of phase B, the rest is phase A.

[0087] Since the polarity of the target product iCDN-N3 after cross-linking becomes smaller, the retention time in the reverse phase chromatographic column is delayed to 22.62 minutes; the main peak (retention time: 22.62 minutes) was prepared and collected for mass spectrometry analysis. The peak with the highest abundance, i.e., [MH]-peak, was observed in the negative ion mode at 1237.1, and the calculated value was 1237.2, which is within the normal error range and can be confirmed as the target product iCDN-N3 ( Figure 2 The concentration was determined using a NanoDrop micro-spectrophotometer and stored in a refrigerator at -80°C.

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

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

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

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

[0092] like Figure 4 As shown, the results showed that free 2′3′-cG s A s Compared with MP-treated cells, iCDN-N3-treated cells showed significantly increased 2'3'-cG s A s MP level.

[0093] 5. iCDN-N3 can increase the relative mRNA content 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 h, followed by the addition of THPP (5 mM) and incubation for another 8 h before analysis. s A s MP (i.e., liposome nanoparticles encapsulating 2'3'-cG s A s MP), with an equimolar concentration of 2'3'-cG s A s Cells were transfected with MP and encapsulated in Lipofectamine 3000 for 5 h (2'3'-cG s A s MP@Lipo) and then incubated in fresh DMEM medium (purchased from Cell Technology (Beijing) Co., Ltd.) for another 8 hours. In addition, a blank group (Blank), a group using iCDN-N3 alone (iCDN-N3), a group using THPP alone (THPP), and a group using free 2'3'-cG s A s The MP group (2'3'-cG s A s MP) was used as a control. Cells from each group were collected to extract RNA for reverse transcription and used for RT-PCR analysis.

[0095] The results are as follows Figure 5 As shown, free 2'3'-cG s As MP slightly induced the expression of IFNβ and CXCL10 in HEK293 cells, which was mainly 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 an approximately 10-fold and 15-fold increase in the expression of IFNβ and CXCL10, respectively, which was significantly higher than that of the positive control 2'3'-cG s A s MP@Lipo delivery of 2'3'-cG s A s The activities of MP were comparable.

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

[0097] CT26 cells were seeded in 6-well plates (400,000 cells per well), treated with iCDN-N3 (2 μM) for 5 h, and then incubated in DMEM medium (purchased from CellTech (Beijing) Co., Ltd.) containing THPP (5 mM) for 8 h. s A s The MP treatment group and the positive control group 2'3'-cG s A s MP@Lipo) was used at the same molar concentration as iCDN-N3. The cells were lysed with RIPA buffer containing protease inhibitors and phosphatase inhibitors to extract total protein.

[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, verifying the activation of the STING signaling pathway at the protein level.

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

[0100] Female Balb / c mice were placed in pathogen-free conditions at 25 ± 2 °C and 40% relative humidity. 4T1 cells were then injected subcutaneously into the mice (1.5 × 10 6 When the tumor volume reaches about 100mm 3The mice were randomly divided into different groups (n=4), namely, 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 used an equal amount of PBS buffer instead of THPP; the THPP group used an equal amount of PBS buffer instead of iCDN-N3; the blank group used an equal amount of PBS buffer instead of iCDN-N3 and THPP.

[0102] At the end of the experiment, mice were killed, and the main organs and tumors were collected and washed with 1× PBS. Tumors were prepared, and the volume was measured and analyzed by RT-PCR. Blood samples were collected and serum was extracted for biochemical index analysis. Tumor volume was measured with a vernier caliper and calculated as follows: tumor volume (mm 3 )=0.5×length×width 2 .

[0103] like Figure 7 As shown, the tumor volume of the iCDN-N3 and THPP co-treated group was reduced by about 70% compared with the blank group. In the group treated with iCDN-N3 or THPP alone, the tumor volume was almost unchanged compared with the blank group. RT-PCR analysis of the tumor was performed, and the results were shown in Figure 8 As shown, significantly increased levels of proinflammatory cytokines including CXCL10, IFNβ, TNFα, IFNγ, and IL6 were detected in tumors of the iCDN-N3 and THPP co-treated group compared with those of the blank group.

[0104] To evaluate the biotoxicity of iCDN-N3 combined with THPP, the body weight of mice was monitored during the treatment period. Fig. 9 As shown in the figure, no significant weight loss occurred in the mice in each group over the treatment period. The biochemical functions of the liver and kidney of the treated mice were analyzed, and the results were as follows: Fig.10 As shown in the figure, there was no significant difference in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), triglyceride (TG), and creatine kinase (CK) between the mice in the iCDN-N3 and THPP co-treated group and those in the blank group.

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

[0106] 1. Synthesis of small molecule compounds with bromine 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) and 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 obtain the product 10 as 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 obtained 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 several times. The organic phases were combined and dried in vacuo to obtain compound 11 without further purification.

[0108] The synthetic route is as follows:

[0109]

[0110] 2. Small molecule compound 11 and 2′3′-cG s A s Cross-linking of MPs

[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 was dissolved in phosphate buffer to obtain a second solution, in which 2′3′-cG s A s The concentration of MP is 100 μM. The two solutions were mixed in a volume ratio of 1:1, stirred at room temperature (30°C) for 48 hours, the reaction solution was collected, vacuum freeze-dried, and the organic solvent was removed. The obtained sample was then redissolved in sterile water and analyzed and purified using high performance liquid chromatography (the purification method is the same as in Example 1). Since the polarity of the target product iCDN-TCO after cross-linking becomes smaller, the retention time in the reverse phase chromatography column is delayed to 21.4 minutes; the main peak was prepared and collected (retention time: 21.4 minutes) and subjected to mass spectrometry analysis. The peak with the highest abundance was observed in the negative ion mode, that is, the [MH]-peak was 1218.7, and the calculated value was 1219.3, which is within the normal error range and can be confirmed as the target product iCDN-TCO ( Fig.11The concentration was determined using a NanoDrop micro-spectrophotometer and stored in a refrigerator at -80°C.

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

[0114] iCDN-TCO (2 μM) was incubated with 3,6-dimethyl-1,2,4,5-tetrazine (Me2Tz, 80 μM) at room temperature for 2 hours, and then the obtained reaction solution was analyzed by high performance liquid chromatography (analysis method was the same as in Example 1). After adding Me2Tz for activation, the retention time of the reaction product was advanced from 21.4 minutes of 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 was observed in the negative ion mode, that is, the [MH]-peak was 705.3 (2′3′-cG s A s The calculated value of [MH]- of MP is 705.1, and the normal error range is 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 shows that iCDN-TCO can be deprotected and release 2′3′-cG under the activation of Me2Tz. s A s MP( Fig.12 ).

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

[0116] Under cell culture conditions of 37°C and 5% carbon dioxide, equal molar concentrations of iCDN-TCO and 2′3′-cG were directly added to HEK293 cells. s A s MP, without transfection, cells were collected and lysed after 5 h of incubation, in which the iCDN-TCO group was deprotected by adding 3,6-dimethyl-1,2,4,5-tetrazine and then measured by the cGAMP ELISA detection kit.

[0117] like Fig.13 As shown, the results showed that free 2′3′-cG s A s Compared with MP-treated cells, iCDN-TCO-treated cells showed significantly increased 2'3'-cG s A s MP level.

[0118] 5. iCDN-TCo can increase the relative mRNA content 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 h, followed by the addition of 3,6-dimethyl-1,2,4,5-tetrazine (80 μM) and incubated for another 8 h before analysis. s A s MP, with equimolar concentrations of 2'3'-cG s A s Cells were transfected with MP and encapsulated in Lipofectamine 3000 for 5 h (2'3'-cG s A s MP@Lipo) and then incubated in fresh DMEM medium (purchased from Cell Technology (Beijing) Co., Ltd.) for another 8 hours. In addition, a blank group (Blank) and a group using iCDN-TCO alone (iCDN-TCO), a group using Me2Tz alone (Me2Tz), and a group using free 2'3'-cG s A s The MP group (2'3'-cG s A s MP) was used as a control. Cells from each group were collected to extract RNA for reverse transcription and used for RT-PCR analysis.

[0120] The results are as follows Fig.14 As shown, free 2'3'-cG s A s MP slightly induced the expression of IFNβ and CXCL10 in HEK293 cells, which was mainly due to its low cell permeability. In cells treated with iCDN-TCO or 3,6-dimethyl-1,2,4,5-tetrazine alone, the changes in IFNβ and CXCL10 expression were very low. In contrast, co-treatment of iCDN-TCO and 3,6-dimethyl-1,2,4,5-tetrazine resulted in approximately 8-fold and 20-fold increases in the expression of IFNβ and CXCL10, respectively, which were significantly higher than those of the positive control 2'3'-cG s A s MP@Lipo is quite good.

[0121] Example 3 Preparation of iCDN-NBs activated by light induced by specific wavelengths and activation of the STING pathway

[0122] 1. Synthesis of small molecule compounds with bromine 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 obtained reaction solution was then extracted and the concentrated organic phase was separated using a silica gel column (petroleum ether / ethyl acetate, volume ratio 4:1) to obtain compound 13 as a light yellow oil (45 mg, yield 78.2%).

[0124] The synthetic route is as follows:

[0125]

[0126] 2. Small molecule compound 13 and 2′3′-cG s A s Cross-linking of MPs

[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 was dissolved in phosphate buffer to obtain a second solution, in which 2′3′-cG s A s The concentration of MP is 100 μM. The two solutions were mixed in a volume ratio of 1:1, stirred at room temperature (30°C) for 48 hours, the reaction solution was collected, vacuum freeze-dried, and the organic solvent was removed. The obtained sample was then redissolved in sterile water and purified using high performance liquid chromatography (the purification method is the same as in Example 1). Since the polarity of the target product iCDN-NB after cross-linking becomes smaller, the retention time in the reverse phase chromatography column is delayed to 17.67 minutes; the main peak (retention time: 17.67 minutes) was prepared and collected and subjected to mass spectrometry analysis. The peak with the highest abundance was observed in the negative ion mode, that is, the [MH]-peak was 1002.9, and the calculated value was 1003.1, which is within the normal error range and can be confirmed as the target product iCDN-NB ( Fig.15 The concentration was determined using a NanoDrop micro-spectrophotometer and stored in a -80°C refrigerator.

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

[0130] iCDN-NB (2 μM) was exposed to 365 nm light for 50 minutes, and the reaction solution was analyzed by HPLC (analysis method was the same as in Example 1) to determine the change in retention time of the target product, and the target peak was analyzed by mass spectrometry. The results showed that after being exposed to 365 nm light activation, the retention time of the reaction product was advanced from 17.67 minutes of 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., [MH]-peak, was 705.2 (2′3′-cG s A s The calculated value of [MH]- of MP is 705.1, and the normal error range is 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, indicating that iCDN-NB can release 2′3′-cG by deprotection under light activation. s A s MP( Fig.16 ).

[0131] 4. iCDN-NB can increase the relative mRNA content 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, then exposed to light at 365 nm for 50 minutes, and then analyzed. In addition, a blank group (Blank), a group using light alone (Blank+Light), and a group using iCDN-NB alone without light (iCDN-NB) were set as controls. Cells in each group were collected to extract RNA for reverse transcription and used for RT-PCR analysis.

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

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

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

[0136] 3-(2,3,5-Trimethyl-1,4-benzoquinone)-3-methylbutanoic 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, and then compound 6 (358 mg, 1.5 eq, 1.5 mmol) was added to the reaction solution. The obtained reaction solution was stirred at room temperature overnight. After the reaction, the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, volume ratio 8:2) to obtain compound 15 as 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 obtained reaction mixture was reacted in an ice bath for 30 minutes and then stirred at room temperature for 3 hours. Saturated sodium bicarbonate solution and dichloromethane in a volume ratio of about 1:1 were added to the reaction solution for extraction. The organic phases obtained after extraction were combined and purified by silica gel column (eluent: petroleum ether / ethyl acetate, volume ratio 9:1) to obtain compound 16 (300 mg, yield 96%).

[0137] The synthetic route is as follows:

[0138]

[0139] 2. Small molecule compound 16 and 2′3′-cG s A s Cross-linking of MPs

[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 was dissolved in phosphate buffer to obtain a second solution, in which 2′3′-cG s A sThe concentration of MP is 100 μM. The two solutions were mixed in a volume ratio of 1:1, stirred at room temperature (30°C) for 48 hours, the reaction solution was collected, vacuum freeze-dried, and the organic solvent was removed. The obtained sample was then redissolved in sterile water and purified by high performance liquid chromatography to obtain iCDN-QP (the purification method is the same as in Example 1). Since the polarity of the target product iCDN-QP after cross-linking becomes smaller, the retention time in the reverse phase chromatography column is delayed to 16.7 minutes; the main peak (retention time: 16.7 minutes) was prepared and collected and subjected to mass spectrometry analysis. The peak with the highest abundance was observed in the negative ion mode, that is, the [MH]-peak was 1381.6, and the calculated value was 1381.4, which is within the normal error range and can be confirmed as the target product iCDN-QP ( Fig.18 The concentration was determined using a NanoDrop micro-spectrophotometer and stored in a -80°C refrigerator.

[0142] 3. In vitro verification of iCDN-QP deprotection and release of 2′3′-cG under NQO1 enzyme activation 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 obtained reaction solution was analyzed by HPLC (the analysis method was the same as in Example 1) for the change in the retention time of the target product, and the target peak was subjected to mass spectrometry analysis. The results showed that after the addition of NQO1 enzyme and reduced coenzyme II (NAD(P)H) for activation, the retention time of the reaction product was advanced from 16.7 minutes of iCDN-QP to 13.37 minutes; the main peak (retention time: 13.37 minutes) was collected and prepared, and the target peak was subjected to mass spectrometry analysis. The peak with the highest abundance was observed in the negative ion mode, namely, the [MH]-peak was 705.2 (2′3′-cG s A s MP's [MH] - The calculated value is 705.1, and the normal error range is 705.1±0.3). It can be considered that the HPLC and mass spectrometry results of the reaction product are consistent with the standard 2′3′-cG s A s MP is consistent, which shows that iCDN-QP can be deprotected and release 2′3′-cG under NQO1 enzyme activation. s A s MP( Fig.19 ).

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

[0145] BMDM, CT26, and 4T1 cells were seeded in 12-well plates (200,000 cells per well), incubated overnight at 37°C, and then treated with iCDN-QP (2 μM) for 8 hours (the above cells contain NQO1 enzyme and reduced coenzyme II (NAD(P)H) and no additional addition is required) before analysis.

[0146] The cells in each group were collected and RNA was extracted for reverse transcription and used for RT-PCR analysis. It can be seen that as the relative content of NOQ1 in BMDM, CT26 and 4T1 cells gradually increased ( Fig. 20 ), iCDN-QP activated endogenous NOQ1 enzyme, and the relative mRNA contents of IFNβ and CXCL10 also increased in turn ( Fig.21 ).

[0147] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to the technical content disclosed above to obtain equivalent or equivalent implementation schemes, which belong to the scope of the present invention.

Claims

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

2. The cyclic dinucleotide derivative according to claim 1 or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt, wherein: R1 and R2 are each independently selected from adenine, guanine, xanthine and hypoxanthine, preferably each independently adenine or guanine.

3. The cyclic dinucleotide derivative according to claim 1 or 2, or a stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt thereof, wherein: R3 and R4 are both hydroxyl groups.

4. A method for preparing a cyclic dinucleotide derivative according to any one of claims 1 to 3 or a stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt thereof, comprising the following steps: The compound represented by formula (II) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt is reacted with the compound Br-LG to obtain the compound represented by formula (I) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt; wherein the compound Br-LG is selected from one or more of the following:

5. The method according to claim 4, wherein: The method comprises the following steps: (1) dissolving the compound Br-LG in a solvent to obtain a first solution; (2) dissolving the compound represented by formula (II) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt in a buffer solution to obtain a second solution; (3) The first solution and the second solution are mixed, and stirred at 5-45°C, preferably 25-35°C, for 24-72 hours, preferably 36-48 hours.

6. The method according to claim 5, wherein: In step (1), the concentration of the compound Br-LG in the first solution is 40-100 mM, preferably 45-55 mM; Preferably, in step (1), the solvent is dimethyl sulfoxide and / or N,N-dimethylformamide.

7. The method according to claim 5 or 6, wherein: In step (2), the concentration of the compound represented by formula (II) or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt in the second solution is 80-150 μM, preferably 95-105 μM; Preferably, in step (2), the buffer is a phosphate buffer; further preferably, the concentration of phosphate in the phosphate buffer is 40-60 mM, preferably 45-55 mM; further preferably, the pH of the phosphate buffer is 6-7; Preferably, in step (3), the volume ratio of the first solution to the second solution is 1:0.5-2, preferably 1:0.8-1.

2.

8. The method according to any one of claims 4 to 7, wherein: The method further comprises: after stirring, freeze-drying and purifying the obtained reaction product in sequence.

9. A pharmaceutical composition for anti-tumor, comprising the cyclic dinucleotide derivative according to any one of claims 1 to 3 or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

10. Use of the cyclic dinucleotide derivative or its stereoisomer, tautomer, hydrate, solvate, nitrogen oxide or pharmaceutically acceptable salt according to any one of claims 1 to 3 in the preparation of an anti-tumor drug; Preferably, the anti-tumor drug is a drug that inhibits tumor growth.

Images