CN114940679B - STING agonist prodrug compound, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a STING agonist prodrug compound and a preparation method thereof, and the molecular structure of the STING agonist prodrug compound is shown as a formula (I). The STING agonist prodrug compound has simple structure and easy preparation, and can be used as a medicine for cancer immune combination therapy. The invention also discloses application of the STING agonist prodrug compound in preparing medicines for cancer immunotherapy, treating obesity, viral infection, liver injury or glycolipid metabolic disorder, and application of the STING agonist prodrug compound in preparing medicines for inhibiting tumor cell growth or killing tumor cells.
Description
Technical Field
The invention relates to the field of chemistry and biological medicine, in particular to a STING agonist prodrug compound, a preparation method and application thereof.
Background
Prodrugs (prodrugs) are compounds which release the active substance in vivo by enzymatic or non-enzymatic action to exert its pharmacological action, which often are novel chemical entities formed by covalent linkage of the active drug (prodrug) to a non-toxic compound. The prodrug should have the following three conditions: (1) According to the molecular property of the medicine with biological activity, chemical modification is carried out according to the treatment requirement; (2) After entering the body, whether the action of enzyme is needed or not, the original medicine molecules are ensured to be recovered; (3) does not exhibit biological activity itself.
Prodrugs are a very important drug design technique. The prodrug technology can effectively change the physicochemical property of the drug, improve the curative effect, improve the pharmacokinetics of the drug, improve the biocompatibility, target the targeting and reduce the toxic and side effects.
Currently, cancer immunotherapy is changing our strategies for treating cancer, and Immune Checkpoint Blocking (ICB) therapy aims at inhibiting immune suppressors activated by immune cells, thereby enhancing the killing ability of immune cells against tumor cells.
The indoleamine 2, 3-dioxygenase (IDO) pathway is used by cancer cells to suppress the immune response of the host in order to facilitate survival, growth, invasion and metastasis of malignant cells. IDO pathways are very active in many cancers, providing a direct defense against T cell attacks. IDO inhibition has shown clinical efficacy and is the mainstay strategy for treating cancer. Because ICB therapy is limited and resistant to patients, immunotherapy is often a combination therapy for better improvement of efficacy and longer duration of benefit.
The interferon gene stimulating factor (STING) plays an important pivotal role in the natural immune response triggered by virus, bacteria and parasite infection, the tumor immune process of the organism and the cell autophagy process; protein synthesis and IFN expression are regulated through self-phosphorylation, ubiquitination and dimerization modification, and play a key role in a plurality of immune links of an organism. Many viruses can cause proliferation of the virus or autoimmune disease by interacting with signal proteins on the cGAS-STING pathway, thereby stimulating the body to produce interferons in an amount unequal to the amount of normal immune response; tumor Cell proliferation activates STING in antigen presenting cells, thereby activating T Cell-mediated adaptive immune processes, exerting antitumor effects (maz, damania b.the cGAS-STING defense pathway and its counteractionby viruses [ J ]. Cell host & microbe,2016,19 (2): 150-158).
Compounds that modulate interferon gene Stimulators (STING) have been an important research topic for cancer and infectious disease treatment and vaccine adjuvants, and STING agonists can increase tumor infiltration of tumor site T cells (TIL) and Dendritic Cells (DC), enhancing killing ability against tumors.
STING agonists have been used in combination therapies with other immunotherapies and have been shown to increase tolerance by IDO inhibitor binding, and therefore, binding of STING agonists to IDO inhibitors may be a potentially effective cancer treatment.
Disclosure of Invention
The invention provides a STING agonist prodrug compound (MSA-NLG), which connects STING agonist MSA-2 and IDO inhibitor NLG-919 through ester group, has the characteristics of easy preparation, high efficiency and the like, and can be used for preparing medicines for treating various cancers.
The technical scheme of the invention is as follows:
a STING agonist prodrug compound, the molecular structure of which is shown in formula (I):
the STING agonist prodrug compound is a prodrug combined by STING agonist (MSA-2) and IDO inhibitor (NLG-919), and can be used as an immunological combination therapy for releasing two drugs in response to esterase to treat various cancers.
The invention also provides a preparation method of the STING agonist prodrug compound, which comprises the following steps:
(1) MSA-2 and NLG-919 are fully dissolved in an organic solvent, then N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DAMP) are added, and stirred at room temperature for reaction;
(2) Removing the organic solvent after the reaction is finished, and purifying the residues by adopting a reverse phase chromatography to obtain the STING agonist prodrug compound;
preferably, in step (1), the molar ratio of MSA-2 to NLG-919 is 1:0.5-2; most preferably 1:1.
Preferably, in step (1), the organic solvent is dichloromethane; the amount of dichloromethane is 1-10mL/mmol based on the amount of MSA-2; most preferably 5mL/mmol.
Preferably, in step (1), the molar ratio of N, N' -dicyclohexylcarbodiimide to MSA-2 is 1-3:1; the molar ratio of 4-dimethylaminopyridine to MSA-2 is 1:5-15.
Most preferably, in step (1), the molar ratio of N, N' -dicyclohexylcarbodiimide to MSA-2 is 2:1; the molar ratio of 4-dimethylaminopyridine to MSA-2 was 1:10.
Preferably, in the step (2), when the residue is purified by reverse phase chromatography, the chromatographic column model is shim-packGISC of 18 mm by 250mm; the mobile phase is a mixture of 4 by volume: 6 ultrapure water: a mixed solvent of acetonitrile; the elution speed is 5mL/min; the column temperature was 30 ℃.
The STING agonist prodrug compound has stronger cancer treatment effect. Based on the above, the invention also provides application of the STING agonist prodrug compound in preparing medicines for cancer immunotherapy, treating obesity, viral infection, liver injury or glycolipid metabolic disorder.
The invention also provides application of the STING agonist prodrug compound in preparing a medicament for inhibiting the growth of tumor cells or killing the tumor cells.
Preferably, the tumor cell is a breast cancer cell, an oral cancer cell, a liver cancer cell, a lung cancer cell, a stomach cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a bladder cancer cell or a prostate cancer cell.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a STING agonist prodrug compound MSA-NLG which has a simple structure and is easy to prepare, and the STING agonist prodrug compound MSA-NLG can be used as a medicine for cancer immune combined treatment. MSA-NLG improves the limitation of a single IDO inhibitor to a certain extent, enhances the immune stimulation response of the STING agonist to the tumor microenvironment, and has good application prospect in the combined immunotherapy administration strategy and prodrug design.
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FIG. 1 is a graph showing the results of the MSA-NLG mass spectrometry performed in example 2;
FIG. 2 shows the induced expression level of IFN- β in mouse macrophages by MSA-NLG;
FIG. 3 shows the change in the cell viability of mouse macrophages under different treatment conditions;
FIG. 4 is a prodrug nanoparticle-activated mouse tumor model anti-tumor immune microenvironment;
FIG. 5 is an anti-tumor activity of prodrugs in a mouse engraftment tumor model.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
EXAMPLE 1 Synthesis of prodrug MSA-NLG
MSA-2 (1 mmol) and NLG-919 (1 mmol) were dissolved in dichloromethane (5 mL), after waiting for sufficient dissolution, N' -Dicyclohexylcarbodiimide (DCC) (2 mmol) and 4-Dimethylaminopyridine (DAMP) (0.1 mmol) were added to the solution, and the solution was stirred at room temperature for reaction for 24 hours. Then, the remaining methylene dichloride in the system was removed by evaporation under reduced pressure, and separation and purification were carried out by reversed phase chromatography (column model is shim-packGISC18 20mm x 250 mm), with ultrapure water in a volume ratio of 4:6: eluting with acetonitrile mixed solvent as mobile phase at eluting speed of 5mL/min and column temperature of 30deg.C, collecting new peak liquid to obtain product MSA-NLG, and determining molecular weight of 559.2276 by LC-MS, wherein the result is shown in figure 1.
Example 2 the STING agonism of mouse macrophages by MSA-NLG was demonstrated by measuring the induced expression of IFN- β in the mouse macrophages (5 h incubation in vitro).
After culturing and processing the mouse macrophages, cell culture supernatants were collected at designated time points and the amount of IFN- β expression was measured using the mouse IFN- β Elisa kit (Multiscience, EK 2236-96). The effect of the prodrug on IFN- β production by mouse macrophages before and after treatment with esterase is shown in FIG. 2. The secretion of IFN- β was induced by the STING agonist MSA-2 alone and by the prodrug treatment of mouse macrophages in the presence of PLE esterase, whereas no increase in IFN- β secretion was observed with the prodrug alone, demonstrating the efficacy and safety of the drug.
Example 3 biocompatibility and effectiveness of prodrugs and their individual components were tested by detecting changes in mouse macrophage cell viability.
After incubation and treatment of mouse macrophages, macrophages were then treated with indicated concentrations of MSA-2, NLG919 and prodrug-loaded nanoparticles and incubation continued for various periods (5 h,24h,48 h). The cells were then washed with PBS and mixed with CCK-8 reagent and incubated in an incubator for 1h. The optical density was measured by an enzyme-labeled instrument (Thermo Scientific) in 3 replicates, and the number of living cells was represented by the average value, and the results were shown in FIG. 3, and A, B, C in FIG. 3 was the result of macrophage culture for 5h,24h,48h, respectively. The results show that the administration did not affect cell viability when observed at early time points, but decreased cell viability was observed for longer culture periods with some toxicity.
Example 4 detection of the modulation of the tumor immune microenvironment by prodrugs by immunofluorescence detection of changes in immune cell populations.
After culturing and treating the different treatment groups, tumor tissues of the different treatment groups were collected, and the change of immune cell population was detected by IF, and the change of immune cell population in TME of the prodrug-coated nanoparticle was detected, and the results were shown in FIG. 4, wherein A, B, C, D in FIG. 4 is CD3, respectively + T cells, CD8 + T cells, CD11 + Dendritic cells, gr-1 + Distribution of inhibitory cells E is a representative picture of tumor TUNEL staining after treatment with different drugs. A, B, C in FIG. 4 shows CD3 in the prodrug-treated group + T cells, CD8 + T cells, CD11 + Dendritic cells were significantly increased and enriched compared to the other treatment and control groups. D in fig. 4 shows that the prodrug reduced the number of inhibitory cellular MDSCs in the tumor microenvironment and E in fig. 4 shows that the tumors of the treated group underwent significant apoptosis compared to the control group.
Example 5 in vivo experiments in mice demonstrate that prodrugs have the effect of inhibiting tumor growth and prolonging survival in mice.
After the mice were randomized into 4 groups, physiological saline, NLG919 (100 ul,10 mM), MSA-2 (100 ul,15 mg/kg) and prodrug-coated nanoparticles (100 ul,15 mg/kg) were injected at the indicated time points by tail vein method, respectively. Tumor size was measured every other day with calipers and tumor volume was calculated according to the formula 0.5 x length x width. In the survival analysis, the tumor volume reached 2000mm 3 Is considered dead. At the end of the follow-up period, mice were euthanized, tumors resected, weighed and analyzed by imaging. The results are shown in fig. 5, wherein a is a schematic diagram of tumor inoculation and treatment scheme of mice, B is a tumor volume growth curve of mice during 12 days of follow-up, C is a tumor growth curve of 4 different tumor groups, each curve represents different mice, D is a representative photograph of anatomical tumors of each group of mice after 12 days of follow-up, and E is a survival curve of each group of tumor-bearing mice. B, C, D in FIG. 5 shows that the tumor growth of each treatment group was significantly inhibited compared to the vehicle-treated group, as seen in tumor volume, with MSA-2 and prodrug NPs treated groups being the most effective. E in FIG. 5 shows that mice treated with MSA-2 and prodrug NPs have an extended survival time compared to the vehicle-treated group.
The foregoing embodiments have described the technical solutions and advantages of the present invention in detail, and it should be understood that the foregoing embodiments are merely illustrative of the present invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like that fall within the principles of the present invention should be included in the scope of the invention.
Claims (9)
1. A STING agonist prodrug compound, characterized in that its molecular structure is represented by formula (I):
2. a method of preparing a STING agonist prodrug compound according to claim 1, comprising the steps of:
(1) MSA-2 and NLG919 are fully dissolved in an organic solvent, then N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DAMP) are added, and stirred at room temperature for reaction;
(2) Removing the organic solvent after the reaction is finished, and purifying the residues by adopting a reverse phase chromatography to obtain the STING agonist prodrug compound;
3. the method of claim 2, wherein in step (1), the molar ratio of MSA-2 to NLG919 is 1:0.5-2.
4. The process according to claim 2, wherein in step (1), the organic solvent is methylene chloride; the amount of methylene dichloride is 1-10mL/mmol based on the amount of MSA-2 material.
5. The process according to claim 2, wherein in step (1), the molar ratio of N, N' -dicyclohexylcarbodiimide to MSA-2 is 1-3:1; the molar ratio of 4-dimethylaminopyridine to MSA-2 is 1:5-15.
6. The preparation method according to claim 2, wherein in the step (2), when the residue is purified by reverse phase chromatography, the column model is shim-packGISC18 mm x 250mm; the mobile phase is a mixture of 4 by volume: 6 ultrapure water: a mixed solvent of acetonitrile; the elution speed is 5mL/min; the column temperature was 30 ℃.
7. Use of a STING agonist prodrug compound according to claim 1 in the preparation of a medicament for the immunotherapy of cancer.
8. Use of a STING agonist prodrug compound according to claim 1 in the preparation of a medicament for inhibiting the growth of or killing tumor cells.
9. The use according to claim 8, wherein the tumor cell is a breast cancer cell, an oral cancer cell, a liver cancer cell, a lung cancer cell, a stomach cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a bladder cancer cell or a prostate cancer cell.
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