3, 5, 6-trimethylbenzofuran-2-ketone derivative and application thereof in STING activation

Abstract

The invention provides a 3, 5, 6-trimethylbenzofuran-2-ketone derivative and application thereof in STING activation, and the derivative has a structure as shown in a formula (I) and a formula (IV). In the prior art, through a strategy of knocking out STIM1 through CRISPR-Cas9, although cGAMP-mediated I-type interferon secretion is improved, intracellular calcium ion concentration is remarkably reduced due to irreversibility of knockout, calcium ion balance is destroyed, and processes of subsequent antigen cross presentation and the like are not facilitated. According to the present invention, the small molecule is developed to achieve the separation of STIM1 and STING, and the intracellular calcium ion steady state is maintained while the STING endoplasmic reticulum retention is removed so as to effectively enhance the tumor treatment effect of the STING agonist, and particularly, compared with the STING agonist previously disclosed by the applicant, the reported compound has characteristics of strong STING sensitivity enhancing ability, good STING sensitivity enhancing effect, and good tumor treatment effect. Meanwhile, good anti-tumor activity and anti-inflammatory activity are achieved, and the clinical immune response rate is expected to be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to a 3,5,6-trimethylbenzofuran-2-one derivative, its preparation method and application. Background Technology

[0002] According to the International Agency for Research on Cancer, cancer is the second leading cause of death worldwide. The pathogenesis of cancer involves not only uncontrolled cell proliferation but also complex immune evasion mechanisms. Research into the interaction between cancer and the immune system has led to cancer immunotherapy becoming a transformative breakthrough in recent years.

[0003] Since its discovery in 2008, the stimulator of interferon genes (STING) has been recognized as a key molecule in the cGAS-STING cytoplasmic DNA sensing pathway, playing a crucial regulatory role in modulating the host immune response. STING is a key signal adaptor protein in the DNA sensing pathway, located at rest in the endoplasmic reticulum membrane. Upon activation by cytoplasmic DNA, it translocates to the endoplasmic reticulum-Golgi interstitial compartment (ERGIC) to recruit TANK-binding kinase 1 (TBK 1) and interferon regulatory factor 3 (IRF 3). IRF 3, after phosphorylation by TBK 1, homodimerizes and translocates to the nucleus to induce the transcription of type I IFN. Given its central role in immunity and its profound impact on cancer biology, STING has garnered significant attention in oncology.

[0004] The transport of STING protein from the endoplasmic reticulum (ER) to the Golgi apparatus is the rate-limiting step in its activation process. Studies have found that stromal interaction molecule 1 (STIM1), located on the ER membrane, can significantly inhibit the transport efficiency of STING to the Golgi apparatus by specifically binding to it. This mechanism directly affects the efficacy of clinical STING agonist therapy, and this inhibitory effect mainly depends on the interaction of their transmembrane domains on the ER membrane. Notably, clinical observation revealed that a patient with a STIM1 loss-of-function mutation exhibited significant activation characteristics of the type I interferon signaling pathway, including upregulation of the expression levels of various cytokines and interferon-stimulated genes. This finding not only validates the regulatory role of STIM1 in the STING pathway but, more importantly, demonstrates that targeted intervention of the STIM1-STING interaction may be an effective strategy to enhance STING pathway activation. Therefore, developing interventions that can specifically block STIM1-mediated STING retention is of significant clinical importance for improving the sensitivity of STING-targeted therapy.

[0005] STIM1 is a Ca containing an EF chiral structure. 2+ This binding protein is distributed throughout the endoplasmic reticulum (ER) when calcium ion concentration is high; however, when the ER calcium pool is depleted, it translocates to the ER-membrane interface, interacts with the Orai1 (calcium release-activated calcium modulator 1) pore subunit of the calcium pool-operated calcium channel, and induces calcium absorption. 2+ Influx. Calcium ions, as important messengers, play multiple roles in tumor immune regulation. They not only participate in key immune steps such as antigen presentation and T cell activation, but are also crucial for the normal function of the STING pathway. When STIM1-mediated calcium channel function is impaired (e.g., through gene knockout), although it may promote the production of type I interferon in the short term, it disrupts intracellular calcium homeostasis, thus inhibiting subsequent immune response cascades, including antigen presentation efficiency and the tumor-killing ability of effector T cells. Therefore, it is necessary to develop small molecules to separate STIM1 and STING, relieving STING endoplasmic reticulum retention while maintaining intracellular calcium homeostasis, thereby effectively enhancing the tumor therapeutic effect of STING agonists. Summary of the Invention

[0006] Existing technologies, such as CRISPR-Cas9 knockout of STIM1, while enhancing cGAMP-mediated type I interferon secretion, suffer from irreversible knockout leading to a significant decrease in intracellular calcium ion concentration, disrupting calcium homeostasis and hindering subsequent antigen cross-presentation processes. This invention develops a small molecule to separate STIM1 from STING, relieving STING's endoplasmic reticulum retention while maintaining intracellular calcium homeostasis, thereby effectively enhancing the tumor therapeutic effect of STING agonists and potentially significantly improving clinical immune response rates. Specifically, this invention provides the application of 3,5,6-trimethylbenzofuran-2-one derivatives in sensitizing STING activation, anti-inflammatory effects, and anti-tumor activity. In particular, compared with the previously reported compound ((7-((3,4-dihydroisoquinoline-2(1H)-yl)sulfonyl)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl)methyl ketone), the compound of the present invention has a structural modification in the tetrahydroisoquinoline moiety. The resulting compound of formula I or formula IV not only exhibits stronger STING sensitizing ability, but also has good antitumor and anti-inflammatory activities. This significant technological advancement is beyond people's imagination.

[0007] A 3,5,6-trimethylbenzofuran-2-one derivative having the structure shown in formula (I) or formula (IV):

[0008] ;

[0009] ;

[0010] Wherein, R1 and R3 are independently selected from substituted or unsubstituted polycyclic heterocyclic groups; R2 and R4 are independently selected from one or more self-selected substituents, wherein the self-selected substituents are selected from one or more of nitro, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, and trifluoromethyl; and Linker is a linking group containing a thioether, amine, amide, or sulfonamide structure.

[0011] In this invention, the substituted or unsubstituted polycyclic heterocyclic group contains at least one element selected from nitrogen, oxygen, or sulfur, wherein the substituent includes C1-C6 alkyl groups; the selected substituent is selected from one or more of nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 haloalkoxy, and trifluoromethyl groups; and the linker is a thioether group, amino group, amide group, or sulfonamide group.

[0012] Furthermore, the substituted or unsubstituted multi-heterocyclic group includes pyridine, imidazole, piperidine, benzylamine, and quinoline; the linker is a thioether group.

[0013] Preferably, the 3,5,6-trimethylbenzofuran-2-one derivative has the following structure:

[0014] , .

[0015] This invention discloses a method for preparing the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives, comprising the following steps: subjecting the raw material of formula (II) to ring cladding and substituent modification to obtain intermediate of formula (III) or intermediate of formula (V); subjecting intermediate of formula (III) or intermediate of formula (V) to oxidation and sulfonamide to obtain 3,5,6-trimethylbenzofuran-2-one derivative of formula (I) or 3,5,6-trimethylbenzofuran-2-one derivative of formula (IV);

[0016] ;

[0017] ;

[0018] .

[0019] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows.

[0020] The application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in STING activation, or the application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in the preparation of STING activating reagents.

[0021] The above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives are used in the sensitization and activation of STING, or the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives are used in the preparation of sensitization and activation reagents for STING.

[0022] The above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives are used in promoting the separation of STIM1 and STING, or the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives are used in the preparation of reagents that promote the separation of STIM1 and STING.

[0023] The above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives are used in reducing the co-localization of STING with the endoplasmic reticulum, relieving STING retention in the endoplasmic reticulum, or promoting the translocation of STING from the endoplasmic reticulum to the Golgi apparatus; or the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives are used in the preparation of reagents that reduce the co-localization of STING with the endoplasmic reticulum, relieve STING retention in the endoplasmic reticulum, or promote the translocation of STING from the endoplasmic reticulum to the Golgi apparatus.

[0024] The application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in the preparation of drugs.

[0025] The application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in the preparation of anti-inflammatory drugs.

[0026] The application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in the preparation of tumor therapeutic drugs.

[0027] The application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in the preparation of reagents to improve the therapeutic effect of tumor drugs.

[0028] The 3,5,6-trimethylbenzofuran-2-one derivatives disclosed in this invention have excellent biological activity, can promote the separation of STIM1 and STING and promote the transport of STING from the endoplasmic reticulum to the Golgi apparatus, and significantly increase the level of IFN-β secreted by STING agonists.

[0029] Therefore, this invention discloses the application of the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives in the treatment of tumors.

[0030] In particular, the 3,5,6-trimethylbenzofuran-2-one derivatives of the present invention can enhance the efficacy of existing STING activators, that is, they can sensitize existing STING activators.

[0031] This invention is the first to discover that 3,5,6-trimethylbenzofuran-2-one derivatives have the function of relieving STING endoplasmic reticulum retention and have a significant sensitizing effect on STING activation, and can be used as STIM1 molecular glue. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the synthesis reaction of the compound in Example 1.

[0033] Figure 2 This is an affinity diagram of the compound in Example 1 for STIM1.

[0034] Figure 3 This is an affinity diagram of the compound in Example 2 for STIM1.

[0035] Figure 4 The effects of different compounds combined with SR-717 on IFN-β secretion. Detailed Implementation

[0036] This invention provides a 3,5,6-trimethylbenzofuran-2-one derivative having the structure shown in formula (I):

[0037] ;

[0038] Furthermore, the 3,5,6-trimethylbenzofuran-2-one derivative is preferably of formula (I-1):

[0039] .

[0040] This invention also discloses a 3,5,6-trimethylbenzofuran-2-one derivative having the structure shown in formula (Ⅳ):

[0041] ;

[0042] Furthermore, the 3,5,6-trimethylbenzofuran-2-one derivative is preferably of formula (Ⅳ-1):

[0043] .

[0044] This invention provides a method for preparing the above-mentioned 3,5,6-trimethylbenzofuran-2-one derivatives, comprising the following steps:

[0045] The present invention obtains intermediates of formula (III) / (V) by cladding and substituent modification of the raw material of formula (II), and then obtains 3,5,6-trimethylbenzofuran-2-one derivatives of formula (I) / (IV) by oxidation and sulfonation.

[0046] ;

[0047] ;

[0048] .

[0049] This invention is the first to discover that compounds of the 3,5,6-trimethylbenzofuran-2-one class possess the ability to relieve STING endoplasmic reticulum retention and have a significant sensitizing effect on STING activation, and can be used as STIM1 molecular gels.

[0050] The following specific experiments illustrate the technological advancements of this invention. The raw materials used are all existing products, and the specific preparation operations, performance tests, and data statistical analyses are all conventional techniques. The animal experiments meet the relevant requirements of Soochow University.

[0051] Example 1: Synthesis of (7-((6-methoxy-1,2,3,4-tetrahydroquinoline-1-yl)sulfonyl)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl)methyl ketone

[0052]

[0053] See Figure 1 The intermediate was obtained by cladding and substituent modification of the raw material, followed by oxidation and sulfonation to yield the compound (7-((3-carbonyl-1,2,3,4-tetrahydroquinoline-1-yl)sulfonyl)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl)methyl ketone, formula (I-1); the specific synthetic method is as follows:

[0054] Under argon atmosphere and at 0 °C, 1-(2-hydroxy-4,5-dimethylphenyl)ethyl-1-one (1640 mg, 10 mmol, 1 equivalent) and ethyl 4-bromoacetoacetate (1.336 mL, 12 mmol, 1.2 equivalent) were added to DMF (100 mL), and the mixture was refluxed at 100 °C for 12 hours. After cooling to room temperature, EtOAc (30 mL) and H2O (50 mL) were added to the solution. The mixture was then poured into a saturated NaHCO3 aqueous solution (30 mL), and the aqueous layer was extracted with DCM (20 mL × 3). The organic layer was collected, washed with a saturated Na2CO3 aqueous solution, dried on Na2SO4, and the solvent was removed under vacuum. The crude product was then purified by column chromatography (AcOEt / cyclohexane, 1 / 100 to 1 / 50) to obtain ethyl 3,5,6-trimethylbenzofuran-2-carboxylate (1612 mg, 6.94 mg). (mmol, 69%), is a white solid;

[0055] At room temperature, LiHMDS (2317 mg, 2 equivalents) was added to a mixture of ethyl 3,5,6-trimethylbenzofuran-2-carboxylate (1612 mg, 1.0 equivalent), piperidine (707.88 mg, 1.2 equivalent), and toluene (300 mL, 15.0 volume). The mixture was stirred for 15 hours. After the reaction was completed, the mixture was poured into 600 mL of H2O at 0 °C. The aqueous layer was then extracted with AcOEt (20 mL × 3). The organic layer was collected, washed with saturated NaCl aqueous solution, dried on Na2SO4, and the solvent was removed under vacuum. The crude product was purified by column chromatography (AcOEt / cyclohexane, 1 / 20) to give piperidin-1-yl(3,5,6-trimethylbenzofuran-2-yl) methyl ketone (813.45 mg) as a white solid.

[0056] The compound piperidin-1-yl(3,5,6-trimethylbenzofuran-2-yl) methyl ketone (813.45 mg, 1.0 equivalent) and NBS (N-bromosuccinimide, 533.94 mg, 1.0 equivalent) were added to 50 mL of CH3CN, and then stirred at 80 °C for 12 hours. A saturated NaCl aqueous solution (30 mL) was then added to the mixture, and the aqueous layer was extracted with DCM (10 mL × 3). The organic layer was collected, washed with a saturated NaCl aqueous solution, dried on Na2SO4, and the solvent was removed under vacuum. The crude product was then purified by column chromatography (AcOEt / cyclohexane, 1 / 100 to 1 / 50) to give (7-bromo-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl) methyl ketone (348 mg), a white solid.

[0057] (7-bromo-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl) methyl ketone (348 mg, 1 equivalent), benzyl thiol (186 mg, 1.5 equivalent), DIPEA (N,N-diisopropylethylamine, 580 mg, 3 eq), methyloxanthracene (Xantphos, 173 mg, 0.2 eq), and Pd2(dba)3 (137 mg, 0.1 eq) were added to 50 mL of Dioxane (1,4-dioxane). The mixture was stirred at 100 °C for 2 h. After the reaction was complete, 30 mL of saturated NaCl aqueous solution was added to the mixture, and DCM (20) was used to control the reaction. The aqueous layer was extracted with 3 ml of the extract, and the organic layer was collected and washed with saturated NaClO4 aqueous solution. The organic layer was dried on Na2SO4 and the solvent was removed under vacuum. The crude product was purified by column chromatography (AcOEt / cyclohexane, 1 / 20 to 1 / 10) to give (7-(benzylthio)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl) methyl ketone (390 mg), which was a white solid.

[0058] NCS (N-chlorosuccinimide, 400 mg, 3 equivalents) was added to a mixed solvent of AcOH / H2O (1:1) for compound (7-(benzylthio)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl) (390 mg, 1 equivalent). The reaction was carried out at room temperature for 2 h. The reaction was quenched with H2O, and the aqueous layer was extracted with DCM (dichloromethane, 10 ml × 3). The organic layer was collected, dried on Na2SO4, and the solvent was removed under vacuum to obtain crude 3,5,6-trimethyl-2-(piperidin-1-formyl)benzofuran-7-sulfonyl chloride as a pale yellow solid. Then, 30 ml of DMF, pyridine (5 equivalents), and 6-methoxy-1,2,3,4-tetrahydroquinoline (1... (equivalent), and then stirred at 35℃ for 3 hours, followed by quenching with H2O, extraction and washing with ethyl acetate and aqueous solution, concentration, column packing and purification to obtain the product (7-((6-methoxy-1,2,3,4-tetrahydroquinoline-1-yl)sulfonyl)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl)methyl ketone (215 mg); its NMR data are as follows: 1H NMR (600 MHz, DMSO-d6) δ 6.74 (dt, J = 2.1, 1.0 Hz, 1H), 6.63 (d, J = 7.5 Hz, 1H), 6.53(dd, J = 7.5, 2.0 Hz, 1H), 3.72 (s, 3H), 3.56 (t, J = 5.3 Hz, 4H), 3.14 (t, J= 5.5 Hz, 2H), 2.82 – 2.76 (m, 2H), 2.51 (s, 3H), 2.45 (s, 3H), 2.34 (s, 3H),1.94 (p, J = 5.6 Hz, 2H), 1.71 (p, J = 5.6 Hz, 4H), 1.56 (q, J = 6.0 Hz, 2H).

[0059] Example 2: Synthesis of (7-((3-carbonyl-1,2,3,4-tetrahydroquinoline-1-yl)sulfonyl)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl) methyl ketone

[0060]

[0061] The synthesis method is the same as in Example 1, using 2,3-dihydro-1H-quinoline-4-one as the starting material, replacing 6-methoxy-1,2,3,4-tetrahydroquinoline in Example 1, and otherwise the same, to obtain (7-((3-carbonyl-1,2,3,4-tetrahydroquinoline-1-yl)sulfonyl)-3,5,6-trimethylbenzofuran-2-yl)(piperidin-1-yl) methyl ketone, formula (Ⅳ-1); NMR data are as follows: 1 H NMR (600MHz, DMSO-d6) δ 7.94 (dd, J = 7.3, 2.0 Hz, 1H), 7.34 (td, J = 7.4, 2.0 Hz, 1H), 6.73 – 6.67 (m, 2H), 3.56 (t, J = 5.3 Hz, 4H), 3.35 (t, J = 5.5 Hz, 2H), 2.78 (t, J = 5.5 Hz, 2H), 2.56 (s, 3H), 2.41 (s, 3H), 2.36 (s, 3H), 1.71 (p,J = 5.6 Hz, 4H), 1.56 (q, J = 6.0 Hz, 2H).

[0062] Example 3: Affinity of compound 3,5,6-trimethylbenzofuran-2-one derivatives to STIM1

[0063] The target protein was first biotinylated: the purified protein (Recombinant Human STIM1, purchased from Wuhan Feien Biotechnology Co., Ltd.) was treated with sulfonyl-NHS-LC-biotin reagent (Thermo Fisher Scientific) at 4°C in the dark for 2 hours. Free biotin was then removed using a desalting column, and the labeling efficiency was verified using the HABA method (target biotin:protein molar ratio ≈ 2:1). The biotinylated protein was immobilized as a probe on the surface of a streptavidin (SA) biosensor and captured in running buffer (1×PBS, 0.1% BSA, 0.02% Tween-20, pH 7.4) until a response value of 1.0–1.5 nm was achieved. After baseline stabilization, the sensor was sequentially immersed in 96-well plates containing gradient concentrations of compounds in PBS solutions (0.1% DMSO): starting at 50 μM, 2-fold serial dilutions were performed (50.0, 25.0, 12.5, 6.25, 3.13, 1.56 μM), with a zero-concentration control (containing only run buffer). Each concentration was used in triplicate. The compound binding phase lasted 300 seconds, followed by 60 seconds of dissociation monitoring in run buffer. Data analysis was performed using Octet software, employing a 1:1 binding model to globally fit binding / dissociation curves and calculate kinetic parameters (kon, koff) and the affinity constant KD.

[0064] Figure 2 and Figure 3 The images show the affinity diagrams of the compounds corresponding to Examples 1 and 2 for STIM1. The experimental results show that the compounds corresponding to Examples 1 and 2 have affinity for STIM1, and the dissociation constants (KD) of the corresponding compounds are 9.81E-07 M and 1.23E-06 M, respectively.

[0065] Example 4: Sensitizing activity of compound 3,5,6-trimethylbenzofuran-2-one derivatives on STING activation

[0066] STING is key to bridging innate and adaptive immunity, promoting interferon secretion for tumor killing. STIM1, on the other hand, can inhibit STING activation, thereby suppressing downstream interferon secretion. Therefore, the sensitizing activity of 3,5,6-trimethylbenzofuran-2-one derivatives against STING activation can be determined by measuring IFN secretion.

[0067] Interferon-β (IFN-β) levels were detected using a competitive ELISA method. First, BMDC cells induced to differentiate by 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) were seeded into 48-well culture plates. 5 µM of the compound and 25 μg / mL of the LSTING agonist SR-717 were added to each well, and the cells were incubated for 12 hours. The supernatant was then collected to detect IFN-β levels. During the assay, pre-coated IFN-β antigen competitively bound to biotinylated antibodies against IFN-β in the sample. After washing, horseradish peroxidase-labeled avidin was added to form an immune complex. Finally, a chromogenic substrate (TMB) was added. TMB turned blue under the catalysis of horseradish peroxidase, turning yellow upon the addition of stop solution. OD values ​​were measured at 450 nm, and the concentration of IFN-β in the sample was calculated by plotting a standard curve.

[0068] Using compound (formula VI) of CN2025109972562 as a reference, the following is an example:

[0069]

[0070] Figure 4 The effects of the compounds corresponding to Formula VI in combination with SR-717 on IFN-β secretion were investigated in Examples 1, 2, and VI. Specifically, compared with the use of the STING agonist SR-717 alone, the two representative compounds I-1 and IV-1 reported in Examples 1 and 2, when combined with the STING agonist SR-717, significantly promoted IFN-β secretion (P ≤ 0.01), and were significantly better than the previous compounds (Formula VI).

[0071] Example 5: Determination of the antitumor activity of compound 3,5,6-trimethylbenzofuran-2-one derivatives

[0072] Mouse melanoma cells B16F10 were selected. Cells in the logarithmic growth phase were digested, counted, and adjusted to a homogeneous and stable state before plating. Cells were seeded at a density of 3000 cells / well in 96-well plates (note: the outer wells were filled with PBS). The plates were incubated overnight in a cell culture incubator. After 24 hours, the cells were treated with fresh culture medium containing different concentrations of the test compound. Experimental groups were set up: a blank control group (solvent control group) and experimental groups. The compound concentrations in the experimental groups were 0.05 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, and 20 μM, respectively. Each group had three auxiliary wells. The positive control was dacarbazine, administered using the same method as the test compound. After 72 hours of treatment, 10 μL of MTT solution was added to each well, and the cells were incubated for another 2 hours. The cells were then analyzed using a microplate reader at 492 nm. The absorbance at a wavelength of nm was used to calculate the cell viability per well. Each experiment was repeated three times to calculate the IC50 value of the drug against B16F10 melanoma cells. The results are shown in Table 1. As can be seen from Table 1, the 3,5,6-trimethylbenzofuran-2-one derivatives reported in this invention exhibit good antitumor activity. The two representative compounds reported in this invention, Formula I-1 and Formula IV-1, both showed significant inhibitory activity against mouse B16F10 melanoma cells, and their inhibitory efficacy was stronger than that of the control compound dacarbazine, unexpectedly significantly superior to the compound (Formula VI) in CN2025109972562.

[0073] Table 1. In vitro antitumor activity of the tested compounds

[0074]

[0075] Example 6: Determination of the anti-inflammatory activity of compound 3,5,6-trimethylbenzofuran-2-one derivatives

[0076] Human monocytes (THP-1) were cultured in RPMI 1640 medium (containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.05 mM β-mercaptoethanol) and induced to differentiate into macrophage-like states for 24 hours with 100 ng / ml phorbol ester (PMA). Before the experiment, cells were seeded into 24-well plates at a density of 1 × 10⁻⁶ cells / well. 6Cells were cultured at 37°C, 5% CO2, and saturated humidity. Before the experiment, the cells were starved for 12 h in serum-free RPMI 1640 medium. Different concentration gradients of the test compound were added to each well (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 25 μM, respectively). After pretreatment for 1 h, 100 ng / ml LPS (lipopolysaccharide) was added and incubated for 24 h. The cell supernatant was collected for the detection of interleukin-6 (IL-6) levels. IL-6 was detected using an enzyme-linked immunosorbent assay (ELISA) kit (purchased from Thermo Fisher Scientific). The procedure was followed according to the kit instructions: IL-6 antibody was coated onto the ELISA plate and incubated overnight at 4°C; after blocking non-specific binding sites, a series of concentrations of IL-6 standards and the supernatant to be tested were added, and the plate was incubated at 37°C for 1 hour; unbound components were washed away, and biotin-labeled IL-6 detection antibody was added, and the plate was incubated at 37°C for 30 minutes; horseradish peroxidase-labeled avidin was then added, and the plate was incubated at 37°C for 15 minutes; after washing, the chromogenic substrate TMB was added, and the plate was incubated in the dark for 10 minutes; the reaction was terminated by adding stop solution. The OD values ​​of each well were measured at 450 nm using a microplate reader. Since IL-6 concentration is directly proportional to OD450, a four-parameter logistic function standard curve was plotted with standard concentration on the x-axis and corresponding OD values ​​on the y-axis. The concentration of IL-6 in the sample was calculated using the standard curve. The experiment included a blank control group (culture medium only), a model group (LPS stimulation, no compound), and a positive control group (dexamethasone, concentration gradient consistent with the test compound, administration method the same as the test compound), with three replicates per group. The experiment was repeated three times, and the average IL-6 concentration of each group was recorded. The IL-6 inhibition rate was calculated, and a four-parameter logistic curve was fitted with compound concentration on the x-axis and IL-6 inhibition rate on the y-axis to calculate the IC50 of the compound on IL-6 secretion from THP-1-derived macrophages. 50 The value was used to assess its anti-inflammatory activity.

[0077] The results are shown in Table 2. The experimental results show that the 3,5,6-trimethylbenzofuran-2-one derivatives reported in this invention have good anti-inflammatory activity. The two representative compounds reported in this invention, Formula I-1 and Formula IV-1, both showed significant anti-inflammatory activity, and their inhibitory efficacy was stronger than that of the control compound dexamethasone and also better than that of compound (Formula VI).

[0078] Table 2. In vitro anti-inflammatory activity of the test compounds

[0079]

[0080] Compared with existing compounds, the compounds of the present invention have the following significant advantages:

[0081] (1) The 3,5,6-trimethylbenzofuran-2-one derivatives reported in this invention have stronger STING sensitizing ability. In particular, the representative compounds I-1 and IV-1 reported in this invention both show stronger STING sensitizing ability than existing compounds. They can be used to prepare STING activating reagents, prepare tumor therapeutic drugs, or prepare reagents to improve the therapeutic effect of tumor drugs.

[0082] (2) The 3,5,6-trimethylbenzofuran-2-one derivatives reported in this invention have good antitumor activity. In particular, in vitro inhibition assays against mouse melanoma cells B16F10 show that the IC50 values ​​of the representative compounds I-1 and IV-1 of this invention are high. 50 The values ​​were 10 μM and 3 μM, respectively, which were superior to the control drug dacarbazine (IC50). 50 =19 μM) and prior compound VI (IC) 50 =25μM), which can be used as a candidate compound for the preparation of drugs for the treatment of related tumor diseases;

[0083] (3) The 3,5,6-trimethylbenzofuran-2-one derivatives reported in this invention have good anti-inflammatory activity. In particular, the representative compounds I-1 and IV-1 of this invention can significantly inhibit the release of IL-6, IC50, and IL-6. 50 The values ​​were 84 nM and 43 nM, respectively, which were superior to the control drug dexamethasone (IC50). 50 =132 nM) and prior compound VI (IC 50 =108 nM), exhibiting significant anti-inflammatory activity.

Claims

1. A 3,5,6-trimethylbenzofuran-2-one derivative having the structure shown in formula (I) or formula (IV): ； ； in, R1 and R3 are independently selected from substituted or unsubstituted polycyclic heterocyclic groups; R2 and R4 are independently selected from one or more self-selected substituents, wherein the self-selected substituents are selected from one or more of nitro, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, and trifluoromethyl; and Linker is a linking group containing a thioether, amine, amide, or sulfonamide structure.

2. The 3,5,6-trimethylbenzofuran-2-one derivative according to claim 1, characterized in that, The substituted or unsubstituted polycyclic heterocyclic group contains at least one element selected from nitrogen, oxygen, or sulfur, wherein the substituent includes C1-C6 alkyl groups; the selected substituent is selected from one or more of nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 haloalkoxy, and trifluoromethyl groups; and the linker is a thioether group, amino group, amide group, or sulfonamide group.

3. The 3,5,6-trimethylbenzofuran-2-one derivative according to claim 2, characterized in that, The substituted or unsubstituted multi-heterocyclic group includes pyridine, imidazole, piperidine, benzylamine, and quinoline; the linker is a thioether group.

4. The 3,5,6-trimethylbenzofuran-2-one derivative according to claim 1, characterized in that, The 3,5,6-trimethylbenzofuran-2-one derivatives have the following structures: 、 。 5. A method for preparing the 3,5,6-trimethylbenzofuran-2-one derivative according to any one of claims 1 to 4, comprising the following steps: The raw material of formula (II) is modified by clasping and substituents to obtain intermediate of formula (III) or intermediate of formula (V); The intermediate of formula (III) or formula (V) is oxidized and sulfonated to obtain 3,5,6-trimethylbenzofuran-2-one derivatives of formula (I) or 3,5,6-trimethylbenzofuran-2-one derivatives of formula (IV); ； ； 。 6. The use of the 3,5,6-trimethylbenzofuran-2-one derivatives according to any one of claims 1 to 4 in the preparation of STING activating agents.

7. The use of the 3,5,6-trimethylbenzofuran-2-one derivatives according to any one of claims 1 to 4 in the preparation of sensitizing STING activating reagents, or in the preparation of reagents that promote the separation of STIM1 and STING.

8. The use of the 3,5,6-trimethylbenzofuran-2-one derivatives according to any one of claims 1 to 4 in the preparation of reagents that reduce the colocalization of STING with the endoplasmic reticulum, relieve STING retention in the endoplasmic reticulum, or promote the translocation of STING from the endoplasmic reticulum to the Golgi apparatus.

9. The use of the 3,5,6-trimethylbenzofuran-2-one derivatives according to any one of claims 1 to 4 in the preparation of pharmaceuticals.

10. The use of the 3,5,6-trimethylbenzofuran-2-one derivatives according to any one of claims 1 to 4 in the preparation of anti-inflammatory drugs; or in the preparation of tumor therapeutic drugs; or in the preparation of reagents for improving the therapeutic effect of tumor drugs.

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