CN116253706B - Targeting Siglec-9-promoted immune cell anti-tumor and antiviral function small molecule inhibitor and application thereof - Google Patents

Abstract

The invention belongs to the field of immunological technology, antitumor drugs and antiviral drugs, relates to a targeting Siglec-9 immune cell promotion antitumor and antiviral function small molecule inhibitor and application thereof, and provides 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene with a structure shown in a formula I]-1,1", 3" -tetraketone compounds and pharmaceutically acceptable salts, stereoisomers, tautomers or prodrug molecules thereof and uses thereof. The compound can be used as a Siglec-9 small molecule inhibitor, promotes the anti-tumor and antiviral functions of immune cells, and realizes the treatment of tumor and HBV infection.

Description

A small molecule targeting Siglec-9 to promote anti-tumor and anti-viral functions of immune cells Inhibitors and their applications

Technical field

The invention belongs to the fields of immunology technology, anti-tumor drugs and anti-viral drugs, and in particular relates to a small molecule inhibitor that promotes immune cell (T, NK cell) function by targeting Siglec-9.

Background technique

The information in this Background section is disclosed solely for the purpose of increasing understanding of the general background of the invention and is not necessarily considered to be an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.

Sialic acid-binding immunoglobulin-like lectin family (Siglec) protein, as a new immunosuppressive molecule, has become a new focus in the study of anti-viral immunity and anti-tumor immunity in recent years. Siglec family proteins are mainly expressed on the surface of lymphocytes and mainly transmit inhibitory signals. Most of their ligands are sialylated glycan proteins. High sialylation of glycoproteins is a characteristic pathological change of various solid tumors and hematological tumors. Sialyl glycans are involved in basic molecular and cell biological processes of cancer, such as cell signaling and communication, tumor cell isolation and invasion, and cell- Matrix interactions, tumor angiogenesis, immune modulation, and metastasis formation. Alterations in sialylation regulate cancer development and progression, serve as important biomarkers, and provide a range of specific targets for therapeutic intervention.

Natural killer cells (NK) are the first identified subtype of innate lymphoid cells (ILC). They can respond to tumor cells and virus-infected cells. Their main function is to kill target cells and produce Cytokines. CD8+T cells are the main effector cells that perform antigen-specific killing of tumor cells. Effectively enhancing the anti-tumor function of CD8+T cells is the key to treating tumors. Tumor cells often use immune checkpoint molecules on the surface of NK and CD8+T to evade the surveillance of these immune cells. These immune checkpoint molecules, including immune checkpoint molecules such as PD-1, TIGIT, CTLA-4 and TIM-3, all There are clear reports that it can inhibit the anti-tumor function of immune cells.

Only Siglec-7 and Siglec-9 are expressed on the surface of NK cells. Both belong to the Siglec family proteins related to CD33. Siglec-9 is selectively expressed in CD56 ^dim NK cells. The juxtamembrane motif ITIM in the intracellular region of Siglec-9 undergoes tyrosine phosphorylation and recruits inhibitory phosphatases, such as tyrosine phosphatase 1 (SHP-1), SHP-2 and SH2 inositol phosphatase (Src Homdogy 2domain containing inositol polyphosphate 5-phosphatase (SHIP), thereby mediating the phosphorylation of downstream tyrosine kinases and subsequently transmitting inhibitory or lethal signals into the cell. Siglec-9 tends to bind to β-galactoside-α-2,3 sialic acid, recruit tyrosine phosphatase SHP-1 and SHP-2, and exert its regulatory functions on a variety of immune cells. Multiple studies have suggested that tumor-derived sialic acid induces the differentiation of monocytes into macrophages through the Siglec-9 signaling pathway. Induces immune cells to release factors related to the tumor microenvironment and disease progression, and induces macrophages to exhibit a tumor-associated macrophage (TAM)-like phenotype. In addition, Siglec-9+ macrophages reduced the secretion of pro-inflammatory factors and increased the expression of PD-L1 and the secretion of IL-10. Siglec-9 participates in the pathological process of sepsis by interacting with TLR4, regulating macrophage polarization and inhibiting neutrophil activation. Siglec-9 expression levels were significantly increased on peripheral CD56 ^dim CD16+ NK cells in cancer patients. In vitro functional experiments show that blocking Siglec-7 or Siglec-9 with Fab fragments can promote the killing effect of NK cells on tumor cells K562. Studies have found that Siglec-9 ligands are significantly highly expressed in human tumor cell lines of different histological types and in tumor biopsies from melanoma patients. Enzymes are used to remove sialoglycan ligands or monoclonal antibodies on tumor cells. , mAb) blocks Siglec-9, significantly enhances NK cell activity, and significantly promotes anti-tumor response. The above studies have clearly shown that Siglec-9 regulates NK cells and plays an important role in anti-tumor immunity. In addition, recent studies have proven that the immunosuppressive receptor Siglec-9 pathway of NK cells in patients with chronic hepatitis B is abnormal. Blocking the Siglec-9 pathway can significantly restore NK cell function in patients with chronic hepatitis B, suggesting that Siglec-9 regulates NK cell function in the chronicity of HBV infection. also plays an important role.

Currently, the only FDA-approved Siglec-targeted therapies are antibody-drug conjugates (ADCs) against CD22 (beboza) and CD33 (gemtuzumab), which use Siglecs as tumor-specific antigens to identify target cells . In addition, a variety of vaccines and monoclonal antibodies (mAbs) targeting glycans or Siglecs have entered Phase II and III clinical trials. However, in view of the inherent shortcomings of monoclonal antibodies, such as lack of oral bioavailability, long-term retention in tissues, poor permeability, immune-related adverse reactions, and high cost, people are increasingly looking forward to using small molecules to replace antibodies to eliminate these shortcomings. Moreover, so far, no small molecule inhibitor of Siglec-9 has been reported.

Contents of the invention

In order to solve the above problems, the present invention provides a 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2' that promotes the anti-tumor function of immune cells by targeting Siglec-9 -Furan-4',2"-indene]-1,1",3,3"-tetraketone small molecule inhibitors and compositions containing the compounds. The present invention also provides the effect of the above compounds on promoting immune cells to resist tumors. Functional activity screening results and their applications.

In order to achieve the above objects, the present invention adopts the following technical solutions:

1. 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”- Tetraketones

The invention discloses a 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1",3 ,3"-tetraketone compounds, or their pharmaceutically acceptable salts, esters or prodrugs, have a structure represented by general formula I:

in,

m is an integer selected from 0, 1 or 2, and each occurrence of R ₁ is independently a C ₁ to C ₂ alkyl group, halogen, hydroxyl, methoxy group, amino group, methylamino group, cyano group, nitro group , halomethyl, formamide, carboxyl, ester group;

R ₂ is: C ₃ to C ₆ cycloalkyl, a benzene ring with 0 to 2 substitutions, a six-membered heterocyclic ring with 0 to 2 substitutions, a five-membered heterocyclic ring with 0 to 2 substitutions, as described The substituents are each independently selected from C ₁ to C ₂ alkyl, halogen, hydroxyl, methoxy, amino, methylamino, cyano, nitro, halomethyl, formamide, carboxyl, and ester groups;

The configuration of the compound is racemate, (3'S,5'R), (3'S,5'S), (3'R,5'R) or (3'R,5'S) configuration.

Preferably according to the present invention,

m is an integer selected from 0, 1 or 2, and each occurrence of R ₁ is independently a C ₁ to C ₂ alkyl group, halogen, hydroxyl, methoxy group, amino group, methylamino group, cyano group, nitro group , halomethyl, formamide, carboxyl;

R ₂ is: a benzene ring with 0 to 2 substitutions, a thiophene ring with 0 to 2 substitutions, and the substituents are each independently selected from C ₁ to C ₂ alkyl groups, halogens, hydroxyl groups, and methoxy groups. , amino, methylamino, cyano, nitro, halomethyl, formamide, carboxyl;

The configuration of the compound is racemate, (3'S,5'R), (3'S,5'S), (3'R,5'R) or (3'R,5'S) configuration.

Further preferred according to the present invention, 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1", 3,3”-tetraketones are one of the following:

The "pharmaceutically acceptable salt" mentioned in the present invention means that within the scope of reliable medical evaluation, the salt of the compound is suitable for contact with the tissues of humans or lower animals without undue toxicity, irritation and allergy. Reactions, etc., have a reasonably reasonable benefit-to-risk ratio, are typically water or oil soluble or dispersible, and are effective for their intended use. Included are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts which are compatible with the chemical properties of the compounds of formula I for their intended use. For a list of suitable salts see S. M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.

"Prodrug" as used in the present invention refers to pharmaceutically acceptable compounds such that the biotransformation products obtained from these compounds are active drugs as defined for the compounds of Formula I.

2. 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”- Promoting anti-tumor function of immune cells and its application of tetraketone compounds

The invention discloses 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1",3,3 "-Activity screening results of tetraketone compounds and their first application as Siglec-9 protein inhibitors. Experiments have proven that the above compounds can promote the anti-tumor function of immune cells by targeting Siglec-9 protein. The present invention also provides the above compounds Application in the preparation of anti-tumor drugs.

1.3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Binding of analogues to Siglec-9 protein

This invention uses virtual screening of compounds that bind to Siglec-9 in the compound library and scores their affinity, integrating affinity scores, rationality of the binding mode, structural characteristics, predicted physical and chemical properties (such as molecular weight, xLogP, etc.) and commercial availability property and other factors, we selected 3',5'-disubstituted-3'H,5'H-bispiro[indene-2,2'-furan-4',2"-indene]-1,1", The 3,3"-tetraketone compound MTX-3937 was further tested for its affinity to Siglec-9 under different concentration gradients. In the biofilm interference technology (BLI) experiment, the compound MTX-3937 showed a concentration-dependent effect on the target protein. Affinity, its KD value was measured to be 3.15μM (Figure 1).

From the above experiments and their results, the following conclusions can be drawn:

Based on computer virtual screening and BLI technology, 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1 was identified ,1”,3,3”-tetraketone compound MTX-3937 has good affinity with Siglec-9 in vitro.

2.3',5'-Disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Compounds regulate the function of NK and T cells through Siglec-9

In order to prove that the compound MTX-3937 can regulate the anti-tumor function of NK cells, the present invention uses MTX-3937 at concentrations of 0, 5, 10 and 20 μM to pretreat the NK92 cell line for 24 hours, and kill K562 cells for 6 hours with different effect-to-target ratios. The results It was shown that compound MTX-3937 can dose-dependently increase the killing ability of NK92 cells against K562 (A in Figure 2). Pretreated NK92 cells were stimulated with PMA/ionomycin, and the secretion of cytokines such as CD107a, TNF-α, and IFN-γ was detected. The results showed that compound MTX-3937 can promote the ability of NK92 to secrete cytokines (B in Figure 2 ).

Peripheral blood from HBV-positive patients and liver cancer patients was collected, and peripheral blood mononuclear cells were extracted. After treatment with 20 μM compound MTX-3937 for 24 hours, they were stimulated with PMA/ionomycin for 6 hours. The results showed that compound MTX-3937 can significantly promote HBV and The ability of peripheral blood NK cells in patients with liver cancer to secrete cytokines (C in Figure 2), and the effect of compound MTX-3937 on cytokine secretion by T cells in peripheral blood of patients (D in Figure 2) was also detected.

Siglec-9 is a cell surface receptor, and activation of its downstream pathways requires binding to ligands. In order to detect whether compound MTX-3937 functions through Siglec-9, the present invention uses Siglec-9 Fc chimeric protein, which is preliminarily combined with compound MTX- 3937 pretreatment blocked the binding of compound MTX-3937 to Siglec-9 on the cell surface, and then tested the ability of NK cells to secrete cytokines. The results showed that adding Siglec-9 Fc offset the promotion of cytokine secretion by compound MTX-3937. function (E in Figure 2).

From the above experiments and their results, the following conclusions can be drawn:

3',5'-Disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone The compound MTX-3937 promotes the function of NK and T cells through Siglec-9.

3.3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Compounds inhibit NK cell apoptosis

In the present invention, compound MTX-3937 is used to treat NK92 cells or patient peripheral blood mononuclear cells for 24 hours, cycloheximide (CHX) is used to induce cell apoptosis for 12 hours, and the positive proportion of AnnexinV in NK cells is detected. The results show that the cells treated with compound MTX-3937 The proportion of cell apoptosis decreased significantly (Figure 3).

From the above experiments and their results, the following conclusions can be drawn:

3',5'-Disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Compound MTX-3937 inhibits NK cell apoptosis.

4.3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Compounds enhance the effect of adoptive reinfusion of NK cells to treat tumors

In the present invention, NSG (nonobese diabetic Prkdc ^em26 IL2rg ^em26 /Gpt) mice are randomly divided into 3 groups. All 3 groups are intraperitoneally injected with HepG2-luciferase cells, and 7 days later, PBS, DMSO-treated NK92 and MTX-3937 are reinfused into the tail vein. NK92 cells were used to dynamically observe tumor growth using small animal in vivo imaging on days 10, 20, and 30, and observe the survival of mice. Statistical results of mouse in vivo imaging showed that mice in the compound MTX-3937-treated group of NK92 cells had slower tumor growth, indicating that NK92 cells successfully inhibited the growth and proliferation of liver cancer (Figure 4, A). Statistics on the survival of the mice showed that the mice in the group treated with compound MTX-3937 had a longer survival period (B in Figure 4).

From the above experiments and their results, the following conclusions can be drawn:

3',5'-Disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone After treating NK cells with the compound MTX-3937, it can significantly promote the anti-tumor ability of NK cells in the body and improve the effect of adoptive reinfusion of NK cells to treat tumors.

5.3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Promoting effect of quasi-compounds on NK cells

The present invention tests more 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1",3, 3"-tetraketone compounds promote the ability of NK92 cells to secrete CD107a. Flow cytometry results show that compounds MTX-3937, MTX-5008, MTX-7030MTX-9045, etc. can significantly promote the level of CD107a secreted by NK92 cells (Figure 5).

From the above experiments and their results, the following conclusions can be drawn:

3',5'-Disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”-tetraketone Compounds can promote the killing function of NK cells.

In short, the present invention discovered for the first time through screening a small molecule inhibitor that acts on the Siglec-9 protein, which can promote the anti-tumor and anti-HBV infection functions of immune cells and can be used to prepare anti-tumor and anti-HBV infection drugs.

Beneficial effects of the invention

(1) Through virtual screening technology, 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1 was obtained ",3,3"-tetraketone compounds, and through biofilm interference technology, the compound MTX-3937 was identified, which has good affinity with Siglec-9 in vitro.

(2) Compound MTX-3937 inhibits NK cell apoptosis.

(3) After treating NK cells with compound MTX-3937, it can significantly promote the anti-tumor ability of NK cells in vivo and improve the effect of adoptive reinfusion of NK cells to treat tumors.

(4) Various 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1",3, 3”-tetraketone compounds can promote the killing function of NK cells.

Description of the drawings

The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

Figure 1. Biofilm interference technology (BLI) determines the affinity of compound MTX-3937 to Siglec-9 protein.

Figure 2. Compound MTX-3937 promotes the cytokine secretion ability and killing function of NK cells through Siglec-9.

Among them, A shows the ability of compound MTX-3937 to promote NK92 cells to kill K562 in vitro; B shows compound MTX-3937 promotes NK92 to secrete cytokines; C shows compound MTX-3937 promotes NK cells in peripheral blood of HBV or HCC patients. Secretion of cytokines; D shows the effect of compound MTX-3937 on the secretion of cytokines by T cells in the patient's peripheral blood, and E shows that using Siglec-9 Fc to block the combination of compound MTX-3937 and cell surface Siglec-9 can restore NK The level of cytokines secreted by cells.

Figure 3. Compound MTX-3937 inhibits NK cell apoptosis.

Figure 4. Compound MTX-3937 promotes the anti-tumor ability of NK cells in vivo.

Among them, A shows that NK92 pretreated with compound MTX-3937 can inhibit the development of liver cancer in mice; B shows that NK92 pretreated with compound MTX-3937 can prolong the survival of tumor-bearing mice.

Figure 5. 3',5'-diphenyl-3'H,5'H-disspiro[indene-2,2'-furan-4',2”-indene]-1,1”,3,3”- Tetraketone compounds promote the ability of NK cells to secrete cytokines.

Detailed ways

It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanation of the present invention, but cannot limit the content of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1: Target protein affinity screening based on biofilm layer interference method.

The binding affinity of compounds to Siglec-9 was determined by using Octet K2 (ForteBio) based on biofilm layer interferometry (BLI). The shaking speed was 1000 rpm, the experimental temperature was set to 30°C, and the buffer was PBS containing 5% DMSO.

(1) Immerse the Super Streptomycin (SSA) sensor in PBS, detect the baseline for 60 seconds, immerse it in 200 μL of 50 μg/mL biotinylated Siglec-9 protein buffer and solidify for 300 seconds. The solidification signal is not less than 4 nm, and detect the baseline after solidification. , measure after the baseline is stable.

(2) Add 200 μL of a certain concentration of the compound to be tested dissolved in the buffer into a black polypropylene 96-well microplate (Greiner 96), and the blank buffer serves as a negative control. A complete detection cycle includes 60 s of baseline detection, 60 s of binding of the compound solution or blank solution to the sensor with immobilized protein, and 90 s of dissociation process. Each concentration is repeated once.

(3) Set up a blank control experiment, that is, repeat the above process using a blank sensor without solidified protein.

(4) Use FortéBio Data Analysis software 9.0 to analyze BLI results. Subtract non-specific and background signals as well as biosensor-induced signal drift by selecting "Dual Reference" mode. Calculate the equilibrium dissociation constant (KD value) based on the ratio of Koff and Kon. Binding kinetics analysis used a 1:1 binding model to calculate KD, K _on , K _off and R ² values.

The results showed that compound MTX-3937 showed concentration-dependent affinity for the target protein, with a measured KD value of 3.15 μM (Figure 1).

Example 2: Extraction of human peripheral blood mononuclear cells.

Take out the human lymphocyte separation tube, balance it to room temperature, and centrifuge at 2000 rpm for 1 minute; slowly add 4 mL of venous whole blood to the separation tube; centrifuge at 2500 rpm for 10 minutes; suck out the white hazy mononuclear cell layer in the middle layer and put it into a new test tube; add 3 mL of PBS buffer and mix Homogenize, centrifuge at 1000rpm for 10min, discard the supernatant to obtain peripheral blood mononuclear cell pellet.

Example 3: In vitro killing function detection of NK cells.

Use human-derived K562 cells as target killing cells, count and take 1×10 ⁷ target cells, and centrifuge at 1200 rpm for 6 minutes to collect cell samples. Resuspend the cell pellet in 1mL PBS buffer, add 1mM CFSE dye, and incubate for 15 minutes at 37°C in the dark for staining. Place the cells on ice for 5 minutes to terminate the chromosome system, add 4 mL of PBS buffer, and centrifuge at 1200 rpm for 6 minutes to wash the cells twice. The cells were resuspended in 100 μM EM-α complete medium and seeded in a 96-well plate. Calculate the corresponding number of NK cells with the ratio of killing effector cells: target cells 10:1, 5:1, and 2.5:1 (NK cells are pre-treated with compound MTX-3937 or DMSO control for 24 hours), and use 100 μL MEM-α complete culture medium Resuspend cells and seed in 96-well plate. After the cell mixture system was incubated at 37°C for 4 hours, cells were collected in flow cytometry tubes, 1 μL of 7-AAD dye was added to each tube, mixed and stained for 5 minutes. The proportion of 7-AAD+ cells in CFSE+ cells was detected by flow cytometry to indicate the killing function of NK cells.

In order to prove that the compound MTX-3937 can regulate the anti-tumor function of NK cells, the present invention uses MTX-3937 at concentrations of 0, 5, 10 and 20 μM to pretreat the NK92 cell line for 24 hours, and kill K562 cells for 6 hours with different effect-to-target ratios. The results It was shown that compound MTX-3937 can dose-dependently increase the killing ability of NK92 cells against K562 (A in Figure 2).

Example 4: Detection of cytokine secretion of NK and T cells.

NK92 cells or extracted peripheral blood mononuclear cells were seeded into a 48-well plate at approximately 5 × 10 ⁵ cells per well, and Siglec-9 Fc (10 μg/mL) or IgG Fc control was incubated with compound MTX-3937 at 37°C. After incubating for 40 minutes, add the cells to the plated cells and treat the cells for 24 hours. PMA (50ng/mL) and ionomycin (1μg/mL) were added 6h before sample collection for stimulation, and BFA (10μg/mL) was added 2h later. Block, add CD107a flow cytometry antibody at the same time, and incubate for another 4 hours; collect cells in a flow cytometry tube, wash once with PBS buffer, centrifuge at 1000 rpm for 5 minutes, dilute to 100 μL, and add surface-labeled flow cytometry antibody PerCp/cy5.5anti -HumanCD3, PE/Cy7anti-HumanCD56 were co-cultured, and placed in the dark at 4°C for 30 minutes; resuspend the cells in 200 μLeBioscience's cell fixative, and fixed in the dark at 4°C for 20 minutes. Add 2 mL of 1xeBioscience's cell transmembrane solution to each tube. Centrifuge at 1200 rpm for 6 minutes, discard the supernatant, wash once with 2 mL of PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 200 μL of fixative to resuspend the cells, fix at 4°C in the dark for 20 min, add 2 mL of membrane-breaking agent, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, leaving about 100 μL of residual liquid. Add anti-intracellular cytokine antibodies (BV421 anti-Human IFN-γ and APC anti-Human TNF-α) for intracellular staining. Place in the dark at 4 degrees for 30 minutes. Add 2 mL PBS to wash the cells. , discard the supernatant, resuspend in 500 μL PBS, and detect by flow cytometry.

Pretreated NK92 cells were stimulated with PMA/ionomycin, and the secretion of cytokines such as CD107a, TNF-α, and IFN-γ was detected. The results showed that compound MTX-3937 can promote the ability of NK92 to secrete cytokines (B in Figure 2 ).

Peripheral blood from HBV-positive patients and liver cancer patients was collected, and peripheral blood mononuclear cells were extracted. After treatment with 20 μM compound MTX-3937 for 24 hours, they were stimulated with PMA/ionomycin for 6 hours. The results showed that compound MTX-3937 can significantly promote HBV and The ability of peripheral blood NK cells in patients with liver cancer to secrete cytokines (C in Figure 2), and the effect of compound MTX-3937 on cytokine secretion by T cells in peripheral blood of patients (D in Figure 2) was also detected.

Siglec-9 is a cell surface receptor, and activation of its downstream pathways requires binding to ligands. In order to detect whether compound MTX-3937 functions through Siglec-9, the present invention uses Siglec-9 Fc chimeric protein, which is preliminarily combined with compound MTX- 3937 pretreatment blocked the binding of compound MTX-3937 to Siglec-9 on the cell surface, and then tested the ability of NK cells to secrete cytokines. The results showed that adding Siglec-9 Fc offset the promotion of cytokine secretion by compound MTX-3937. function (E in Figure 2).

Example 5: NK cell apoptosis detection.

NK cells were seeded into cell plates as above, pretreated with 0, 5, 10 and 20 μM compound MTX-3937 for 24 hours, and CHX was added to induce apoptosis for 12 hours. Collect cells in a flow tube, add PBS, centrifuge at 1200 rpm for 6 min, discard the supernatant, add 200 μL 1×AnnexinV Binding buffer, add AnnexinV antibody, incubate at room temperature for 15 min, and detect with flow cytometry.

Use compound MTX-3937 to treat NK92 cells or patient peripheral blood mononuclear cells for 24 hours, use cycloheximide (CHX) to induce cell apoptosis for 12 hours, and detect the proportion of AnnexinV-positive cells in NK cells. The results show that the cells treated with compound MTX-3937 undergo apoptosis. The proportion dropped significantly (Figure 3).

Example 6: Animal experiment.

A large number of human liver cancer cell line HepG2-luciferase cells were expanded in vitro. After the cell number was sufficient, count and prepare cell suspension (2×10 ⁷ /mL), and inject them into NSG mice (NOD-Prkdc ^em26 IL2rg ^em26 /Gpt). Inject 500 μL cell suspension/animal intraperitoneally. Seven days later, 2×10 ⁵ NK92 cells were injected into the tail vein of the mice (NK92 cells were pretreated with DMSO or compound MTX-3937 for 24 h), and 1×10 ⁵ U IL-2 was intraperitoneally injected every other day to maintain the viability of NK92 cells. On days 10, 20 and 30, the fluorescence signals in the mice were photographed using a small animal in vivo three-dimensional optical imaging analysis system, the signal levels were recorded and analyzed, and the survival curve of the mice was recorded.

In the present invention, NSG (nonobese diabetic Prkdc ^em26 IL2rg ^em26 /Gpt) mice are randomly divided into 3 groups. All 3 groups are intraperitoneally injected with HepG2-luciferase cells, and 7 days later, PBS, DMSO-treated NK92 and MTX-3937 are reinfused into the tail vein. NK92 cells were used to dynamically observe tumor growth using small animal in vivo imaging on days 10, 20, and 30, and observe the survival of mice. Statistical results of mouse in vivo imaging showed that mice in the compound MTX-3937-treated group of NK92 cells had slower tumor growth, indicating that NK92 cells successfully inhibited the growth and proliferation of liver cancer (Figure 4, A). Statistics on the survival of the mice showed that the mice in the group treated with compound MTX-3937 had a longer survival period (B in Figure 4).

Example 7: 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1",3,3 "-The ability of tetraketone compounds to promote the secretion of CD107a by NK92 cells

The present invention tests more 3',5'-disubstituted-3'H,5'H-disspiro[indene-2,2'-furan-4',2"-indene]-1,1",3, The ability of 3"-tetraketone compounds to promote the secretion of CD107a by NK92 cells was tested in the same way as in Example 4. Flow cytometry results showed that compounds MTX-3937, MTX-5008, MTX-7030, MTX-9045, etc. could significantly promote the secretion of CD107a by NK92 cells. level (Figure 5).

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (2)

1. A 3',5'-disubstituted- 3'H , 5'H -disspiro[indene-2,2'-furan-4',2''-indene]-1,1'',3 , the application of 3''-tetraketone compounds or their pharmaceutically acceptable salts or their stereoisomers or their tautomers or their prodrug molecules in the preparation of drugs for preventing or treating HBV infection, and its characteristics That is, the compound has a structure represented by general formula I: in, m is an integer selected from 0, 1 or 2, and R ₁ is independently methyl or halogen each time it appears; R ₂ is: a benzene ring with 0 to 2 substitutions, a thiophene ring with 0 to 2 substitutions, and the substituents are each independently selected from C ₁ to C ₂ alkyl groups, halogens, and nitro groups; The configuration of the compound is racemate, (3 'S , 5'R ), (3 'S , 5 'S ), ( 3'R , 5'R ) or ( 3'R , 5 'S ) configuration. 2. Application as claimed in claim 1, characterized in that it is one of the compounds with the following structure: The configuration of the compound is racemate, (3 'S , 5'R ), (3 'S , 5 'S ), ( 3'R , 5'R ) or ( 3'R , 5 'S ) configuration.