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

Targeting Siglec-9-promoted immune cell anti-tumor and antiviral function small molecule inhibitor and application thereof Download PDF

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CN116253706B
CN116253706B CN202211674354.5A CN202211674354A CN116253706B CN 116253706 B CN116253706 B CN 116253706B CN 202211674354 A CN202211674354 A CN 202211674354A CN 116253706 B CN116253706 B CN 116253706B
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siglec
indene
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CN116253706A (en
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马春红
刘新泳
肖榕
田野
武专昌
梁晓红
高立芬
李春阳
张继伟
赵地
李娜
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Shandong University
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    • C07—ORGANIC CHEMISTRY
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    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/94—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom spiro-condensed with carbocyclic rings or ring systems, e.g. griseofulvins
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
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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

Targeting Siglec-9-promoted immune cell anti-tumor and antiviral function small molecule inhibitor and application thereof
Technical Field
The invention belongs to the fields of immunological technology, antitumor drugs and antiviral drugs, and particularly relates to a small molecule inhibitor for promoting immune cell (T, NK cells) function by targeting Siglec-9.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Sialic acid-binding immunoglobulin-like lectin family (Siglec) proteins have become a new hot spot for studying antiviral immunity and antitumor immunity in recent years as a new immunosuppressive molecule. The Siglec family proteins are mainly expressed on lymphocyte surfaces and mainly transmit inhibitory signals, and the ligands thereof are mostly sialylated glycan proteins. The high sialylation of glycoproteins is a characteristic pathological change in a variety of solid tumors, hematological tumors, and sialylated glycans are involved in fundamental molecular and cellular biological processes of carcinogenesis, such as cell signaling and communication, tumor cell separation and invasion, cell-matrix interactions, tumor angiogenesis, immunomodulation, and metastasis formation. Alterations in sialylation regulate the development and progression of cancer, act as important biomarkers, and provide a range of specific targets for therapeutic intervention.
Natural killer cells (Natural killer cell, NK) are the first identified subset of intrinsic lymphocytes (Innate lymphoid cell, ILC) that respond to tumor cells and virus-infected cells, and function primarily to kill target cells and produce cytokines. Cd8+ T cells are the primary effector cells that perform antigen-specific killing of tumor cells, and effective enhancement of cd8+ T cell antitumor function is critical for treatment of tumors. Tumor cells often evade the monitoring of these immune cells by using immune checkpoint molecules on NK and CD8+ T surfaces, which have been specifically reported to inhibit the anti-tumor function of immune cells, including PD-1, TIGIT, CTLA-4, TIM-3, and the like.
NK cell surfaces are only Siglec-7 and Siglec-9 expressed, both belonging to the CD 33-related Siglec family of proteins, where Siglec-9 is at CD56 dim Selective expression in NK cells. The Siglec-9 intracellular region membrane-proximal motif ITIM undergoes tyrosine phosphorylation, recruiting inhibitory phosphatases such as tyrosine phosphatase 1 (SHP-1), SHP-2 and SH2 inositol phosphatases (SHIP) to mediate downstream tyrosine kinase phosphorylation and in turn deliver inhibitory or lethal signals into the cell. Siglec-9 tends to bind to beta-galactoside-alpha-2, 3 sialic acid, recruits tyrosine phosphatases SHP-1 and SHP-2, and exerts its regulatory function on a variety of immune cells. Several studies suggest that tumor-derived sialic acid induces monocyte differentiation into macrophages through the Siglec-9 signaling pathway. Immune cells are induced to release factors associated with tumor microenvironment and disease progression, and macrophages are induced to exhibit a tumor-associated macrophage (TAM) like phenotype. In addition, siglec-9+ macrophages reduce the secretion of pro-inflammatory factors, increasing the expression of PD-L1 and secretion of IL-10. Siglec-9 participates in sepsis pathology by interacting with TLR4, regulates macrophage polarization, and inhibits neutrophil activation. Peripheral CD56 of cancer patient dim Siglec-9 expression levels on CD16+ NK cells were significantly increased. In vitro functional experiments show that blocking Siglec-7 or Siglec-9 by Fab fragments can promote the killing effect of NK cells on tumor cells K562. The study found that the ligand of Siglec-9 was significantly highly expressed in human tumor cell lines of different histological types and in tumor biopsies of melanoma patients, and that the sialoglycan ligand or monoclonal antibody (MonocThe lon anti-ibody, mAb) blocks Siglec-9, significantly enhances NK cell activity, and significantly promotes anti-tumor responses. The above studies have clearly shown that Siglec-9 regulates NK cells, playing an important role in anti-tumor immunity. In addition, recent researches prove that the immune suppression receptor Siglec-9 channel of NK cells of patients with chronic hepatitis B is abnormal, and blocking the Siglec-9 channel obviously restores the NK cell function of the patients with chronic hepatitis B, which suggests that the Siglec-9 regulation of the NK cell function plays an important role in the chronicity of HBV infection.
Currently, the only FDA approved Siglec targeted therapies are antibody-drug conjugates (ADCs) to CD22 (Bei Bosa) and CD33 (gemtuzumab), which use Siglecs as tumor-specific antigens to recognize target cells. In addition, there are a variety of vaccines and monoclonal antibodies (monoclonal antibody, mAbs) targeting glycans or Siglecs that have entered phase II, III clinical trials. However, in view of the inherent disadvantages of monoclonal antibodies, such as lack of oral bioavailability, prolonged residence in tissues, poor permeability, immune-related adverse reactions, and high cost, there is an increasing desire to eliminate these disadvantages by using small molecules instead of antibodies. Furthermore, no Siglec-9 small molecule inhibitors have been reported so far.
Disclosure of Invention
In order to solve the problems, the invention provides a 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone small molecule inhibitor for promoting the anti-tumor function of immune cells by targeting Siglec-9, a composition containing the compound, and a screening result of the compound for promoting the anti-tumor function activity of immune cells and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
1.3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound
The invention discloses a 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound or pharmaceutically acceptable salt, ester or prodrug thereof, which has a structure shown in a general formula I:
wherein,
m is selected from integers of 0, 1 or 2, R 1 Each occurrence is independently C 1 ～C 2 Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;
R 2 the method comprises the following steps: c (C) 3 ～C 6 Cycloalkyl, benzene ring with 0 to 2 substituents, six membered heterocycle with 0 to 2 substituents, five membered heterocycle with 0 to 2 substituents, said substituents each being independently selected from C 1 ～C 2 Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;
the compound is in the racemate, (3's, 5' r), (3's, 5's), (3 'r,5' r) or (3 'r, 5's) configuration.
According to a preferred embodiment of the present invention,
m is selected from integers of 0, 1 or 2, R 1 Each occurrence is independently C 1 ～C 2 Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl;
R 2 the method comprises the following steps: a benzene ring having 0 to 2 substitutions, a thiophene ring having 0 to 2 substitutions, each of the substituents being independently selected from C 1 ～C 2 Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl;
the compound is in the racemate, (3's, 5' r), (3's, 5's), (3 'r,5' r) or (3 'r, 5's) configuration.
According to a further preferred embodiment of the invention, the 3',5' -disubstituted-3 'H,5' H-dispiro [ indene-2, 2 '-furan-4', 2 "-indene ] -1,1",3 "-tetraketone compound is one of the following:
by "pharmaceutically acceptable salts" as used herein is meant salts of the compounds which are suitable for contact with the tissues of humans or lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, generally water or oil soluble or dispersible, and effective for their intended use, within the scope of sound medical evaluation. Including pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, are contemplated herein and are compatible with the chemical nature of the compounds of formula I. A list of suitable salts is found in S.M. Birge et al, J.Pharm.Sci., pages 1977,66,1-19.
"prodrugs" as used herein refers to pharmaceutically acceptable compounds such that the resulting bioconversion products of such compounds are active agents as defined for the compounds of formula I.
2.3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound with immune cell anti-tumor function promoting function and application thereof
The invention discloses an activity screening result of 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compounds and first application of the activity screening result as a Siglec-9 protein inhibitor. Experiments prove that the compounds can promote the anti-tumor function of immune cells by targeting Siglec-9 protein. The invention also provides application of the compound in preparing antitumor drugs.
1.3',5' -disubstituted-3 'H,5' H-dispiro [ indene-2, 2 '-furan-4', 2 "-indene ] -1,1",3 "-tetraketone binding to Siglec-9 protein
According to the invention, through virtually screening the compounds combined with Siglec-9 in a compound library and scoring the affinity thereof, comprehensive affinity scoring, rationality of a combination mode, structural characteristics, predicted physicochemical properties (such as molecular weight, xLogP and the like), commercial accessibility and other factors are utilized, 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', and 3,3' -tetraketone compounds MTX-3937 are selected to further determine the affinity with Siglec-9 under different concentration gradients. In the biofilm interference technique (BLI) experiment, compound MTX-3937 showed a concentration-dependent affinity for the target protein, with a KD of 3.15. Mu.M (FIG. 1).
From the above experiments and their results, the following conclusions can be drawn:
based on the computer virtual screening and BLI technology, 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound MTX-3937 has good affinity with Siglec-9 in vitro.
2.3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound controls NK and T cell functions through Siglec-9
In order to prove that the compound MTX-3937 can regulate the anti-tumor function of NK cells, the NK92 cell line is pretreated for 24 hours by using MTX-3937 with concentrations of 0,5, 10 and 20 mu M, K562 cells are killed for 6 hours with different effective target ratios, and the result shows that the compound MTX-3937 can improve the killing capacity of the NK92 cells on the K562 in a dose-dependent manner (A in figure 2). Stimulation of pretreated NK92 cells with PMA/ionomycin examined secretion of cytokines such as CD107a, TNF- α and IFN- γ revealed that compound MTX-3937 could promote NK92 secretion (FIG. 2B).
Peripheral blood of HBV positive patients and liver cancer patients was collected, and peripheral blood mononuclear cells thereof were extracted, and after 24 hours treatment with 20. Mu.M compound MTX-3937, stimulation with PMA/ionomycin was performed for 6 hours, which revealed that compound MTX-3937 can significantly promote the ability of HBV and NK cells of peripheral blood of liver cancer patients to secrete cytokines (C in FIG. 2), while the effect of compound MTX-3937 on the secretion of peripheral blood T-cell cytokines of patients was examined (D in FIG. 2).
Siglec-9 was used as a cell surface receptor, the downstream pathway was required to be activated by binding to a ligand, and in order to examine whether or not the compound MTX-3937 functions through Siglec-9, the present invention used a chimeric protein of Siglec-9 Fc, pre-treated with the compound MTX-3937, blocked the binding of the compound MTX-3937 to the cell surface Siglec-9, and then examined the ability of NK cells to secrete cytokines, and the results showed that the promoting effect of the compound MTX-3937 on cytokine secretion was counteracted by the addition of Siglec-9 Fc (E in FIG. 2).
From the above experiments and their results, the following conclusions can be drawn:
3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone MTX-3937 promotes the function of NK and T cells through Siglec-9.
3,3', 5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compounds for inhibiting NK cell apoptosis
In the present invention, NK92 cells or peripheral blood mononuclear cells of patients were treated with the compound MTX-3937 for 24 hours, apoptosis was induced with Cycloheximide (CHX) for 12 hours, and the positive proportion of NK cells Annexin V was detected, which revealed that the proportion of apoptosis treated with the compound MTX-3937 was significantly decreased (FIG. 3).
From the above experiments and their results, the following conclusions can be drawn:
3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone MTX-3937 inhibits NK apoptosis.
4.3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound enhances the effect of adoptive feedback NK cells in treating tumors
The invention uses NSG (nonobese diabetic Prkdc) em26 IL2rg em26 Per Gpt) mice were randomly divided into 3 groups, 3 groups were intraperitoneally injected with HepG2-luciferase cells, and after 7 days, PBS, DMSO-treated NK92 and MTX-3937-treated NK92 cells were returned to the tail vein, respectively, and tumor growth was dynamically observed by live animal imaging for 10, 20, 30 days, and survival of the mice was observed. The results of in vivo imaging statistics of mice showed that the tumor growth of mice treated with the reinfusion compound MTX-3937 in group NK92 cells was slow, indicating that NK92 cells successfully inhibited the growth and proliferation of liver cancer (A in FIG. 4). Survival of mice was counted and found to be the survival period of mice in the feedback compound MTX-3937 treated groupLonger (B in fig. 4).
From the above experiments and their results, the following conclusions can be drawn:
after NK cells are treated by 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound MTX-3937, the anti-tumor capability of the NK cells in vivo can be obviously promoted, and the effect of treating tumors by adoptive feedback of the NK cells is improved.
5.3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone Compounds promotion of NK cells
The invention tests the ability of more 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compounds to promote NK92 cells to secrete CD107 a. The flow results showed that the compounds MTX-3937, MTX-5008, MTX-7030MTX-9045, etc. can significantly promote the level of NK92 cells secreting CD107a (FIG. 5).
From the above experiments and their results, the following conclusions can be drawn:
3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compounds can promote the killing function of NK cells.
In a word, the invention discovers the small molecular inhibitor acting on the Siglec-9 protein for the first time through screening, can promote the anti-tumor and anti-HBV infection functions of immune cells, and can be used for preparing anti-tumor and anti-HBV infection medicines.
The beneficial effects of the invention are that
(1) The 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound is obtained by a virtual screening technology, and the compound MTX-3937 is identified by a biological membrane interference technology, so that the compound has good affinity with Siglec-9 in vitro.
(2) The compound MTX-3937 inhibited NK cell apoptosis.
(3) After NK cells are treated by the compound MTX-3937, the anti-tumor capability of the NK cells in vivo can be obviously promoted, and the effect of treating tumors by adoptively reinfusion of the NK cells is improved.
(4) Various 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compounds can promote the killing function of NK cells.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 biological membrane interferometry (BLI) determines the affinity of the compound MTX-3937 for the Siglec-9 protein.
FIG. 2. Compound MTX-3937 promotes NK cell cytokine secretion and killing function by Siglec-9.
Wherein, A shows the ability of the compound MTX-3937 to promote NK92 cells to kill K562 in vitro; b shows that the compound MTX-3937 promotes NK92 to secrete cytokines; c shows that the compound MTX-3937 promotes the secretion of cytokines by peripheral blood NK cells of HBV or HCC patients; d shows the effect of compound MTX-3937 on the secretion of peripheral blood T-cell cytokines by the patient, and E shows that blocking the binding of compound MTX-3937 to cell surface Siglec-9 using Siglec-9 Fc restores NK cell secretion cytokine levels.
FIG. 3 Compound MTX-3937 inhibits NK cell apoptosis.
FIG. 4 the compound MTX-3937 promotes the anti-tumor ability of NK cells in vivo.
Wherein, A shows that the NK92 pretreated by the reinfusion compound MTX-3937 can inhibit the development of liver cancer in the mouse body; b shows that the NK92 pretreated with the reinfusion compound MTX-3937 can prolong the survival of tumor-bearing mice.
FIG. 5.3',5' -diphenyl-3 'H,5' H-dispiro [ indene-2, 2 '-furan-4', 2 "-indene ] -1,1",3 "-tetraketone compounds promote the ability of NK cells to secrete cytokines.
Detailed Description
It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention, but is not intended to limit the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as 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 interferometry.
Based on biological membrane layer interferometry (BLI), the binding affinity of compounds to Siglec-9 was determined by using Octet K2 (forteBio). The shaking speed was 1000rpm, the experimental temperature was set at 30℃and the buffer was PBS containing 5% DMSO.
(1) A Super Streptomycin (SSA) sensor is immersed in PBS, a baseline is detected for 60 seconds, immersed in 200 mu L of 50 mu g/mL biotinylated Siglec-9 protein buffer solution and cured for 300 seconds, a curing signal is not less than 4nm, the baseline is detected after curing, and the baseline is stable and then measured.
(2) 200. Mu.L of a test compound dissolved in a buffer was added to a black polypropylene 96-well microplate (Greiner 96), and a blank buffer was used as a negative control. One complete detection cycle included a baseline detection of 60s, binding of a 60s compound solution or blank solution to the sensor of the cured protein, and dissociation of 90s, each concentration repeated once.
(3) A blank control experiment was set up, i.e. the procedure was repeated using a blank sensor without immobilized protein.
(4) BLI results were analyzed using Fort Bio Data Analysis software 9.0. By selecting the "dual reference" mode, the non-specific signal and the background signal are subtracted from the biosensor-induced signal drift. The equilibrium dissociation constant (KD value) is calculated from the ratio of Koff to Kon. The binding kinetics analysis adopts a 1:1 binding model to calculate KD, K on ，K off And R is 2 Values.
The results showed that compound MTX-3937 showed a concentration-dependent affinity for the target protein, with a KD of 3.15. Mu.M (FIG. 1).
Example 2: and (5) extracting mononuclear cells of human peripheral blood.
Taking out the human lymphocyte separation tube, balancing to room temperature, and centrifuging at 2000rpm for 1min; 4mL of venous blood whole blood is slowly added into the separating tube; centrifuging at 2500rpm for 10min; sucking out the middle white vaporific mononuclear cell layer, and putting into a new test tube; adding 3mL PBS buffer solution, mixing, centrifuging at 1000rpm for 10min, and discarding the supernatant to obtain peripheral blood mononuclear cell precipitate.
Example 3: and (5) detecting the in-vitro killing function of NK cells.
Human K562 cells were used as killer target cells, and 1X 10 cells were counted and collected 7 The target cells were centrifuged at 1200rpm for 6 minutes to collect a cell sample. Cell pellet was resuspended in 1mL PBS buffer, added 1mM CFSE dye, and incubated at 37 ℃ for 15min in the dark for staining. Cells were placed on ice for 5 minutes, the staining system was terminated, 4mL of PBS buffer was added, and the cells were washed twice by centrifugation at 1200rpm for 6 minutes. Cells were resuspended in 100 μLMEM- α complete medium and plated in 96-well plates. Respectively killing effector cells: target cell ratio 10:1,5:1,2.5:1 the corresponding number of NK cells (NK cells were pre-treated with compound MTX-3937 or DMSO control for 24 h), resuspended cells in 100. Mu.L MEM-alpha complete medium, seeded in 96-well plates. After incubating the cell mixture at 37℃for 4 hours, the cells were collected in flow tubes, 1. Mu.L of 7-AAD dye was added to each tube, and after mixing, stained for 5 minutes. The proportion of 7-AAD+ cells in CFSE+ cells was examined by flow cytometry to show NK cell killing function.
In order to prove that the compound MTX-3937 can regulate the anti-tumor function of NK cells, the NK92 cell line is pretreated for 24 hours by using MTX-3937 with concentrations of 0,5, 10 and 20 mu M, K562 cells are killed for 6 hours with different effective target ratios, and the result shows that the compound MTX-3937 can improve the killing capacity of the NK92 cells on the K562 in a dose-dependent manner (A in figure 2).
Example 4: NK and T cell cytokine secretion detection.
NK92 cells or extracted peripheral blood mononuclear cells were seeded in 48-well plates, about 5X 10 5 Cells per well, after incubating Siglec-9 Fc (10 μg/mL) or IgG Fc control with compound MTX-3937 in a 37 ℃ incubator for 40 min, cells were treated with the plated cells for 24h, stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) 6h before collection, blocked with BFA (10 μg/mL) after 2h, while CD107a flow antibody was added, and incubated for another 4 h; collecting cells in a flow tube, washing with PBS buffer solution once, centrifuging at 1000rpm for 5min, fixing volume to 100 μL, adding surface-labeled flow antibody PerCp/cy5.5anti-HumanCD3 and PE/Cy7anti-HumanCD56, co-culturing, and placing in dark place at 4deg.CPlacing for 30min; cells were resuspended using 200. Mu.L of eBioscience company's cell fixative, fixed at 4℃for 20min in the absence of light, 2mL of 1xeBioscience company's cell-penetrating solution was added to each tube, centrifuged at 1200rpm for 6min, the supernatant was discarded, washed once with 2mL of PBS, centrifuged at 1000rpm for 5min, the supernatant was discarded, 200. Mu.L of fixative was added to resuspend cells, fixed at 4℃for 20min in the absence of light, 2mL of membrane breaker was added, centrifuged at 1000rpm for 5min, the supernatant was discarded, about 100. Mu.L of residual liquid was left, intracellular staining was performed with BV421anti-Human IFN-. Gamma.and APC anti-Human TNF-. Alpha.) by adding anti-intracellular cytokine antibody, standing at 4℃for 30min in the absence of light, washing cells by adding 2mL of PBS, resuspension with 500. Mu.L of PBS after discarding the supernatant, and flow cytometry.
Stimulation of pretreated NK92 cells with PMA/ionomycin examined secretion of cytokines such as CD107a, TNF- α and IFN- γ revealed that compound MTX-3937 could promote NK92 secretion (FIG. 2B).
Peripheral blood of HBV positive patients and liver cancer patients was collected, and peripheral blood mononuclear cells thereof were extracted, and after 24 hours treatment with 20. Mu.M compound MTX-3937, stimulation with PMA/ionomycin was performed for 6 hours, which revealed that compound MTX-3937 can significantly promote the ability of HBV and NK cells of peripheral blood of liver cancer patients to secrete cytokines (C in FIG. 2), while the effect of compound MTX-3937 on the secretion of peripheral blood T-cell cytokines of patients was examined (D in FIG. 2).
Siglec-9 was used as a cell surface receptor, the downstream pathway was required to be activated by binding to a ligand, and in order to examine whether or not the compound MTX-3937 functions through Siglec-9, the present invention used a chimeric protein of Siglec-9 Fc, pre-treated with the compound MTX-3937, blocked the binding of the compound MTX-3937 to the cell surface Siglec-9, and then examined the ability of NK cells to secrete cytokines, and the results showed that the promoting effect of the compound MTX-3937 on cytokine secretion was counteracted by the addition of Siglec-9 Fc (E in FIG. 2).
Example 5: NK cell apoptosis detection.
NK cells were seeded onto cell plates as above, pretreated with 0,5, 10 and 20. Mu.M compound MTX-3937 for 24h, and apoptosis induced by CHX addition for 12h. Cells were collected in a flow tube, centrifuged at 1200rpm for 6min with PBS, the supernatant was discarded, 200. Mu.L of 1X AnnexinV Binding buffer was added, annexin V antibody was added, incubated at room temperature for 15min, and flow cytometry was used for detection.
Treatment of NK92 cells or patient peripheral blood mononuclear cells with Compound MTX-3937 for 24h, induction of apoptosis with Cycloheximide (CHX) for 12h, and detection of the proportion of NK cells AnnexinV positive cells revealed a significant decrease in the proportion of apoptosis with Compound MTX-3937 (FIG. 3).
Example 6: and (5) animal experiments.
In vitro large scale amplification of human liver cancer cell line HepG2-luciferase cells, after sufficient cell number expansion, cell suspension (2×10 7 Per mL), in NSG mice (NOD-Prkdc em26 IL2rg em26 Per Gpt) was intraperitoneally injected with 500. Mu.L of cell suspension per unit. After 7 days, 2X 10 mice were injected i.v. at the tail 5 NK92 cells (pretreatment of NK92 cells with DMSO or Compound MTX-3937 for 24 h) were intraperitoneally injected 1X 10 every other day 5 U IL-2 maintains NK92 cell viability. In vivo fluorescence signals of mice were photographed by a three-dimensional optical imaging analysis system of living animals of the mice at 10, 20 and 30 days, respectively, signal levels were recorded and analyzed, and survival curves of the mice were recorded.
The invention uses NSG (nonobese diabetic Prkdc) em26 IL2rg em26 Per Gpt) mice were randomly divided into 3 groups, 3 groups were intraperitoneally injected with HepG2-luciferase cells, and after 7 days, PBS, DMSO-treated NK92 and MTX-3937-treated NK92 cells were returned to the tail vein, respectively, and tumor growth was dynamically observed by live animal imaging for 10, 20, 30 days, and survival of the mice was observed. The results of in vivo imaging statistics of mice showed that the tumor growth of mice treated with the reinfusion compound MTX-3937 in group NK92 cells was slow, indicating that NK92 cells successfully inhibited the growth and proliferation of liver cancer (A in FIG. 4). Mice survived were counted and the reinfusion compound MTX-3937 treated mice were found to have longer survival (B in FIG. 4).
Example 7:3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compound capable of promoting NK92 cell to secrete CD107a
The invention tests the ability of more 3',5' -disubstituted-3 ' H,5' H-dispiro [ indene-2, 2' -furan-4 ',2' -indene ] -1,1', 3' -tetraketone compounds to promote NK92 cells to secrete CD107a, and the test method is the same as example 4. The flow results showed that compounds MTX-3937, MTX-5008, MTX-7030, MTX-9045, etc. can significantly promote NK92 cells to secrete CD107a (FIG. 5).
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1.3',5' -disubstituted-3 'H,5'H-dispiro [ indene-2, 2 '-furan-4', 2'' -indene]-1,1", 3" -tetraketone compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof or tautomers thereof or prodrug molecules thereof for the preparation of a medicament for the prevention or treatment of HBV infection, characterized in that said compounds have the structure represented by general formula I:
wherein,
m is selected from integers of 0, 1 or 2, R 1 Each occurrence is independently methyl, halogen;
R 2 the method comprises the following steps: a benzene ring having 0 to 2 substitutions, a thiophene ring having 0 to 2 substitutions, each of the substituents being independently selected from C 1 ~C 2 Alkyl, halogen, nitro;
the compound is in the form of racemate, (3'S, 5'R)、(3'S, 5'S)、(3'R, 5'R) Or (3'R, 5'S) Configuration.
2. Use according to claim 1, characterized in that it is one of the compounds having the following structure:
the compound is in the form of racemate, (3'S, 5'R)、(3'S, 5'S)、(3'R, 5'R) Or (3'R, 5'S) Configuration.
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