Divalent metal cationic compounds, methods of making and using the same

Abstract

The present disclosure provides a divalent metal cation compound, a preparation method and application thereof, and belongs to the field of medical treatment. The compound has a structural formula shown in formula (I), wherein R1 is selected from a single bond, a substituted or non-substituted C 1‑5 chain alkylenyl, a substituted or non-substituted C 6‑12 arylenyl; X is selected from O, S and NH; R2 is selected from a C 1‑30 hydrophobic alkylenyl or a polyether, wherein the number average molecular weight of the polyether is 200-2500. The divalent metal cation compound of the present disclosure can bind to phosphatidylserine on the surface of tumor cells, promote the maturation of dendritic cells and inhibit the expression of PD-L1, and can be applied to the preparation of immunomodulatory and antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of medical treatment, in particular to a divalent metal cation compound, a preparation method thereof and an application thereof in a drug for inhibiting PD-L1 and realizing immune anti-tumor effect. BACKGROUND

[0002] Cancer is the leading cause of death in the global population, which seriously hinders the extension of human life expectancy. Anti-programmed death-ligand 1 (PD-L1) / programmed cell death-1 (PD-1) checkpoint blockade therapy can promote T cell-mediated immune surveillance against tumor cells, which significantly changes the treatment prospect of advanced cancer.

[0003] In the related art, the main problems of immune checkpoint therapy are: low response rate, many patients are not sensitive to immune checkpoint therapy; lack of enough tumor-specific antigens, leading to lack of tumor-reactive T cells; after receiving immune checkpoint therapy, the expression of PD-L1 in some patients is up-regulated, leading to drug resistance, thereby reducing the treatment effect and still having the risk of metastasis and recurrence; binding with normal cell surface PD-L1 triggers some adverse reactions, leading to toxic side effects. To solve these problems, it is necessary to develop new and efficient targeting cancer cell mechanisms to reduce toxic side effects on normal tissues; develop mechanisms to deeply reduce the expression of PD-L1 and construct efficient anti-tumor immune drugs. This aspect of research has very good academic research value and also has good social demand. SUMMARY

[0004] Therefore, the present disclosure proposes to construct an organic metal complex having specific binding interaction with phosphatidylserine on the membrane surface of tumor cells, which can selectively bind with phosphatidylserine on the membrane of cancer cells to realize precise targeting of tumors; secondly, by disturbing the structure of the plasma membrane, a large amount of immunogenic substances are induced to be released from cancer cells, DC maturation and antigen presentation are promoted, the tumor "desert type" immunosuppressive microenvironment is improved, and the infiltration and activity of T cells are enhanced; finally, the phosphatidylserine signal is shielded to block the interaction between it and the receptor, the downstream AKT signaling pathway is inhibited to activate, and the expression of PD-L1 is deeply down-regulated, which will not produce drug resistance compared with the commonly used PD-L1 antibody, does not depend on the antibody to reverse the negative feedback mechanism of tumor immunity, reactivates T cell recognition, and thus effectively kills tumor cells.

[0005] One object of the present disclosure is to provide a divalent metal cation compound.

[0006] Another object of the present disclosure is to provide a preparation method of the above-mentioned divalent metal cation compound.

[0007] Still another object of the present disclosure is to provide an application of the above-mentioned divalent metal cation compound.

[0008] The above object of the present disclosure is achieved by the following technical solutions.

[0009] According to an embodiment of one aspect of the present disclosure, there is provided a divalent metal cation compound of Formula I, M is selected from divalent transition metal elements; R1 is selected from a single bond, substituted or unsubstituted C 1-5 chained alkylene, substituted or unsubstituted C 6-12 arylene; X is selected from O, S and NH; R2 is selected from C 1-30 hydrophobic alkyl or polyether, wherein the number average molecular weight of the polyether is 200-2500.

[0010]

[0011] In some embodiments, the divalent transition metal M comprises Ni, Cu, Zn, Cd, Pb, Co, Fe, Ir or Ti.

[0012] In some embodiments, R1 is selected from a single bond, substituted or unsubstituted C 1-5 chained alkylene, substituted or unsubstituted C 6-12 arylene, preferably R1 is a single bond or C 1-5 chained alkylene.

[0013] In some embodiments, R1 is selected from one of the following structures:

[0014]

[0015] In some embodiments, R2 is selected from C 1-16 alkyl or polyethylene glycol chain, for example C 1-8 alkyl.

[0016] In some embodiments, R2 is selected from C 1-30 alkyl or C 2-30 alkenyl, for example C 1-20 alkyl or C 2-20 alkenyl, for example C 1-16 alkyl or C 2-16 alkenyl, for example C 1-8 alkyl or C 2-8 alkenyl.

[0017] In some embodiments, R2 is selected from:

[0018]

[0019] In some embodiments, the divalent metal cation compound has an affinity for the phosphatidylserine on the surface of cancer cells, and the binding affinity is 2.41-10.6 x 10 -6M, and the binding thereof is effective to shield phosphatidylserine signals.

[0020] According to embodiments of another aspect of the present disclosure, a method for preparing a divalent metal cation compound having immunomodulatory and anti-tumor effects shown in formula I is provided, comprising:

[0021] (1) reacting compound (A) and compound (B) in an organic solvent to obtain compound (C);

[0022]

[0023] (2) reacting compound (C) with a divalent metal salt in an organic solvent to obtain the above-mentioned divalent metal cation compound of formula I.

[0024]

[0025] In some embodiments, the hydrophobic or polyether structure of formula R2-X-H includes the following structure:

[0026]

[0027] In some embodiments, the divalent metal salt includes at least one of a nickel salt, a copper salt, a zinc salt, a cadmium salt, a lead salt, a cobalt salt, an iron salt, an iridium salt, or a titanium salt.

[0028] In some embodiments, the reaction in step (1) is carried out in a solution of dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). In some embodiments, the reaction temperature in step (1) is 20-50°C, and the reaction time is 2 hours to 24 hours.

[0029] In some embodiments, the reaction in step (1) is further preferred to be carried out in the presence of dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) and in an inert atmosphere; the reaction temperature in step (1) is 20-50°C, and the reaction time is 4 hours to 12 hours.

[0030] In some embodiments, the reaction temperature in step (2) is 20-40°C, and the reaction time is 1 hour to 24 hours.

[0031] According to embodiments of another aspect of the present disclosure, a use of the above-mentioned divalent metal cation compound in the preparation of a medicament for immunomodulation and anti-tumor is provided.

[0032] In some embodiments, the metal cation compound described above can target the phosphatidylserine on the surface of cancer cells as an immunomodulatory and anti-tumor drug, and has cancer cell selectivity. It shows tumor cell inhibition in vitro and in vivo, with an IC50 of 18.65-37.92 μM for cancer cell lines, and experiments in vivo and in vitro show that it has the function of activating immune cells. The drug described above can block the interaction between phosphatidylserine and phosphatidylserine receptors, inhibit the activation of the downstream AKT signaling pathway, and inhibit the expression of PD-L1. The drug described above has the potential to inhibit the expression of PD-L1, and has the function of immunomodulation and anti-tumor effect.

[0033] Based on the above technical solutions, the bivalent metal cation compound, its preparation method and application provided by the present disclosure have one or some of the following beneficial effects:

[0034] Phosphatidylserine is an important component of human cells and is usually present on the inner side of the cell membrane. However, in the tumor microenvironment, such as hypoxia, low pH, and calcium ion homeostasis disorder, the exposure of phosphatidylserine on the outer leaflet of cancer cells is further enhanced. The externalized phosphatidylserine not only changes the biochemical and physical properties of the plasma membrane, but also initiates a series of interactions between endogenous extracellular proteins and receptors on adjacent cells, forming an immunosuppressive microenvironment and promoting tumor growth and metastasis. The bivalent metal cation compound of the present disclosure can form a strong coordination with the phosphatidylserine on the surface of cancer cells, while there is almost no phosphatidylserine on the outer membrane of normal cells, so the metal cation compound can effectively target tumors. Because it disrupts the plasma membrane structure of cancer cells, it releases tumor-associated antigens and adjuvants, stimulates the maturation of dendritic cells, and improves the tumor immunosuppressive microenvironment. After blocking the interaction between phosphatidylserine and its receptors, it further blocks the downstream AKT signal phosphorylation, reduces the expression of PD-L1, reverses the immune negative feedback mechanism, activates cytotoxic T lymphocytes to recognize and eliminate cancer cells, and effectively achieves tumor immunotherapy.

[0035] The metal cation compound of the present disclosure can target tumors and reduce toxic side effects on normal tissues. The metal cation compound has a simple structure and has great potential for clinical transformation. Compared with traditional immune checkpoint antibody drugs, this new type of anti-tumor immune drug directly reduces the expression of PD-L1 from the source, does not produce antibody treatment resistance, and can inhibit tumor metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0036] The present disclosure will be described in further detail below with reference to the accompanying drawings.

[0037] Figure 1 A synthesis diagram of a bivalent metal cation compound according to an embodiment of the present disclosure is schematically shown;

[0038] Figure 2 Illustrative of the nuclear magnetic resonance hydrogen spectrum of the divalent metal cation compound precursor in Embodiment 1 of the present disclosure is shown;

[0039] Figure 3 Illustrative of the evaluation diagram of specific binding of the divalent metal cation compound to phosphatidylserine (PS) in Embodiment 1 of the present disclosure is shown, wherein a to c are the endothermic amount of the divalent copper metal ion compound, the divalent zinc metal ion compound and the divalent nickel metal ion compound binding to PS / PC over time, d to f are the enthalpy value of the divalent copper metal ion compound, the divalent zinc metal ion compound and the divalent nickel metal ion compound binding to PS / PC over time;

[0040] Figure 4 Illustrative of the scanning electron microscope (SEM) diagram of the cancer cells after treatment with the divalent metal cation compound in Embodiment 1 of the present disclosure is shown, wherein a is the SEM diagram of the control group of cancer cells, b is a magnified view of a, c is the SEM diagram of the cancer cells in the 30 μM concentration group, d is a magnified view of c, e is the SEM diagram of the cancer cells in the 60 μM concentration group, and f is a magnified view of e;

[0041] Figure 5 Illustrative of the release effect diagram of immunogenic substances of the cancer cells after treatment with the divalent metal cation compound in Embodiment 1 of the present disclosure is shown, wherein a to c are the cell ATP change results of cancer cells CT26, B16F10 and 4T1, respectively, and d to f are the release fold change results of extracellular HMGB1 of cancer cells CT26, B16F10 and 4T1, respectively;

[0042] Figure 6 Illustrative of the in vitro anti-tumor effect diagram of the divalent metal cation compound in Embodiment 1 of the present disclosure is shown, wherein a to d are the cytotoxicity results of cancer cells CT26, A549, B16F10 and 4T1, respectively, and e to h are the cytotoxicity results of normal cells L929, 3T3, MCF-10 and LO2, respectively;

[0043] Figure 7 Illustrative of the evaluation diagram of the in vitro reduction of PD-L1 expression of the divalent metal cation compound in Embodiment 1 of the present disclosure is shown, wherein a is the Western blotting analysis result of PD-L1 protein in CT26 cells, and b is the Western blotting analysis result of PD-L1 protein in 4T1 cells;

[0044] Figure 8Figures 1-1 to 1-4 schematically show the in vivo anti-tumor immunotherapy effect of the divalent metal cation compound in Example 1 of the present disclosure, wherein a is the change curve of the tumor volume of the mice, b is the change curve of the body weight of the mice, c is the change curve of the survival rate of the mice, d is the flow cytometry analysis histogram of the mature dendritic cells, e and f are the statistics of the proportion of mature dendritic cells in total dendritic cells, respectively, g is the T cell immunohistochemical staining diagram, h is the statistics of the proportion of CD8 + T cells in total T cells, i is the statistics of the proportion of CD4 + T cells in total T cells;

[0045] Figure 9 Figure 2-1 schematically shows the nuclear magnetic resonance hydrogen spectrum of the metal cation compound precursor in Example 2 of the present disclosure; and

[0046] Figure 10 Figure 2-2 schematically shows the evaluation diagram of the specific binding of the divalent metal cation compound to phosphatidylserine in Example 2 of the present disclosure. DETAILED DESCRIPTION

[0047] Extracellular phosphatidylserine not only changes the biochemical and physical properties of the plasma membrane, but also initiates interactions between endogenous extracellular proteins and receptors on adjacent cells, forms an immunosuppressive microenvironment, and promotes the growth and metastasis of tumors. In the process of implementing the present disclosure, it is found that the hydrophilic part formed by the coordination of terpyridine and divalent transition metal ions can specifically bind to the phosphatidylserine on the surface of cancer cells, thereby realizing the targeting of tumors.

[0048] Therefore, the present disclosure constructs an organometallic complex that has a specific binding interaction with phosphatidylserine on the surface of tumor cell membranes, which includes the above-mentioned hydrophilic part and a hydrophobic chain segment or a hydrophilic chain segment connected to the hydrophilic part. Through its specific binding interaction with phosphatidylserine, the maturation of dendritic cells can be promoted and the expression of PD-L1 can be reduced, thereby realizing the immunotherapy of tumors.

[0049] In order to make the objects, technical solutions and advantages of the present disclosure clearer, the present disclosure is further described in detail below with reference to specific embodiments and the accompanying drawings.

[0050] In the embodiments of the present disclosure, a divalent metal cation compound of formula I is provided, M is selected from divalent transition metal elements; R1 is selected from a single bond, a substituted or unsubstituted C 1-5 chain alkylenyl, a substituted or unsubstituted C 6-12 arylenyl; X is selected from O, S and NH; R2 is selected from C 1-30 hydrophobic alkyl or polyether, wherein the number average molecular weight of the polyether is 200-2500.

[0051]

[0052] In the embodiments of this disclosure, the divalent metal cation compound as shown in Formula 1 consists of a hydrophilic head and a hydrophilic or hydrophobic tail. The hydrophilic head is formed by the coordination of a terpyridine moiety with a divalent transition metal ion, which can effectively coordinate and bind to phosphatidylserine, thereby disrupting the plasma membrane structure, inducing cancer cells to activate and release a large amount of immunogenic substances, further shielding phosphatidylserine signaling, and downregulating PD-L1 expression. The hydrophilic or hydrophobic tail is formed by a hydrophobic chain hydrocarbon group or polyether, which plays a shielding role against phosphatidylserine, blocking the interaction between phosphatidylserine and the receptor, and inhibiting the activation of the downstream AKT signaling pathway.

[0053] In the embodiments of this disclosure, R2 is selected from C. 1-16 R2 is a chain hydrocarbon group or a polyethylene glycol chain, preferably, R2 is selected from C 1-8 Chain hydrocarbon group.

[0054] In the embodiments of this disclosure, R2 is selected from C. 1-30 Alkyl or C 2-30 Alkenyl group, preferably, R2 is selected from C 1-20 Alkyl or C 2-20 Alkenyl group, preferably, R2 is selected from C 1-16 Alkyl or C 2-16 Alkenyl group, preferably, R2 is selected from C 1-8 Alkyl or C 2-8 Alkenyl, more preferably, R2 is selected from one of the following structures:

[0055]

[0056] In the embodiments of this disclosure, when R2 is a hydrophobic chain such as alkyl or alkenyl, it can block the interaction between phosphatidylserine and the phosphatidylserine receptor, shield the signal of phosphatidylserine, inhibit the activation of the downstream AKT signaling pathway, and thus inhibit PD-L1 expression; when R2 is a polyethylene glycol chain, the signal shielding effect can be further enhanced.

[0057] In the embodiments of this disclosure, R1 is selected from single bonds, phenylene, or biphenylene. During the screening of R1, it was found that R1 is selected from one of the following structures, which makes the structure of the divalent metal cation compound more stable and improves its coordination with phosphatidylserine:

[0058]

[0059] In embodiments of the present disclosure, M is selected from Ni, Cu, Zn, Cd, Pb, Co, Fe, Ir or Ti, more preferably Cu; the divalent metal cation compound has affinity with phosphatidylserine on the surface of cancer cells, and the binding affinity constant of the two is 2.41-10.6 x 10 -6 M, through the combination of the two, can effectively shield the interaction of phosphatidylserine with phosphatidylserine receptors.

[0060] According to embodiments of the present disclosure, a method for preparing the divalent metal cation compound with immunomodulatory and anti-tumor effects shown in formula I is also provided, comprising:

[0061] (1) reacting compound (A) and compound (B) in an organic solvent to obtain compound (C);

[0062]

[0063] (2) reacting compound (C) with a divalent metal salt in an organic solvent to obtain the above-mentioned divalent metal cation compound of formula I.

[0064]

[0065] According to embodiments of the present disclosure, through the coordination of the terpyridine ring with the divalent transition metal ion, a head with hydrophilicity is formed; through condensation reaction, a tail with hydrophilicity or hydrophobicity is connected to the hydrophilic head. Figure 1 A synthesis schematic diagram of a divalent metal cation compound according to embodiments of the present disclosure is schematically shown. In some specific embodiments, the synthesis process of a specific metal cation compound of formula I is as shown in Figure 1 .

[0066] In some embodiments of the present disclosure, the reaction in step (1) is carried out in a solution of dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The reaction temperature in step (1) is 20-50°C, for example, 30°C, 35°C, 40°C, 45°C; the reaction time is 2 hours to 24 hours, for example, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours.

[0067] In some embodiments of the present disclosure, the reaction in step (1) is further preferably carried out in the presence of dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) and in an inert atmosphere; the reaction temperature in step (1) is 20-50°C, and the reaction time is 2 hours to 24 hours.

[0068] In some embodiments of the present disclosure, the reaction temperature in step (2) is 20-40℃, for example, 25℃, 30℃, 35℃; the reaction time is 1 hour to 24 hours, for example, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours.

[0069] According to embodiments of the present disclosure, the organic solvent in steps (1) and (2) is independently selected from chloroform, dichloromethane, ethyl acetate, methanol, ethanol, toluene, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, and any combination thereof.

[0070] In some embodiments of the present disclosure, the divalent metal salt comprises at least one of nickel salt, copper salt, zinc salt, cadmium salt, lead salt, cobalt salt, iron salt, iridium salt, or titanium salt, for example, nickel chloride, nickel sulfate, nickel nitrate, copper chloride, copper sulfate, copper nitrate, zinc chloride, zinc sulfate, zinc nitrate, cadmium chloride, cadmium sulfate, cadmium nitrate, lead chloride, lead sulfate, lead nitrate, cobalt chloride, cobalt sulfate, cobalt nitrate, ferrous chloride, ferrous sulfate, ferrous nitrate, iridium chloride, iridium sulfate, iridium nitrate, titanium chloride, titanium sulfate, titanium nitrate, and any combination thereof. Preferably, the divalent metal salt is zinc nitrate, cobalt nitrate, or copper nitrate.

[0071] According to embodiments of the present disclosure, there is also provided a use of the above-mentioned divalent metal cation compound in the preparation of a medicament for immunomodulation and anti-tumor.

[0072] In some embodiments of the present disclosure, the divalent metal cation compound can target tumors and reduce toxic side effects on normal tissues. The metal cation compound has a simple structure and has great potential for clinical transformation.

[0073] According to embodiments of the present disclosure, the divalent metal cation compound provided by the present disclosure specifically binds to phosphatidylserine on the surface of tumor cells, and is suitable for inhibiting the activation of the downstream AKT signaling pathway to inhibit the expression of PD-L1.

[0074] In some embodiments of the present disclosure, compared with traditional immune checkpoint antibody drugs, this new type of anti-tumor immune drug can directly reduce the expression of PD-L1 from the source, does not produce antibody treatment resistance, and can inhibit tumor metastasis, effectively achieving tumor immunotherapy.

[0075] The present disclosure is further illustrated by the following examples. In the following detailed description, numerous specific details are set forth in order to provide a thorough explanation of the embodiments of the present disclosure. It will be apparent, however, that one or more embodiments can be practiced without these specific details. In other instances, well-known methods have not been described in detail in order to not unnecessarily obscure the embodiments presented herein. Also, well-known methods can be combined in any combination, without conflict, into other workable embodiments.

[0076] It should be noted that the following specific examples are only illustrative, and the scope of protection of the present disclosure is not limited thereto. The reagents used in the examples were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., and the cells were purchased from Wuhan Punsun Life Science and Technology Co., Ltd. The methods used in the following examples, such as immunofluorescence staining, are well-known in the art and can be described by textbooks or related literature, and will not be repeated here.

[0077] Example 1

[0078] The method for preparing the divalent metal cation compound comprises the following steps:

[0079] (1) In a 100 mL flask, add terpyridine (100 mg, 0.36 mmol), dicyclohexyl carbodiimide (DCC) (112 mg, 0.54 mmol) and 4-dimethylaminopyridine (DMAP) (13 mg, 0.11 mmol) and dissolve in 5 mL of DMF solution. Farnesol (53 mg, 0.24 mmol) is dissolved in DMF.

[0080] (2) The farnesol solution is added to the 5 mL DMF solution using a constant pressure dropping funnel within 30 min, and the reaction is carried out at 20°C overnight. After stirring overnight, the mixture is cooled and the solvent is removed under vacuum. The crude product is purified by column chromatography (dichloromethane:methanol = 15:1) to obtain a metal cation compound precursor in the form of a white solid. Figure 2 The nuclear magnetic resonance hydrogen spectrum of the metal cation compound precursor in this Example 1 is schematically shown as shown in Figure 2 , and the molecular formula of the compound precursor can be obtained.

[0081] (3) 0.21 mmol of the metal cation compound precursor is dissolved in methanol solution, and copper nitrate (0.21 mmol), zinc nitrate (0.21 mmol) or nickel nitrate (0.21 mmol) is added respectively. The mixed solution is ultrasonically treated at 20-35°C for 60 min, and then the solvent is removed under vacuum, and finally dried to obtain the divalent metal cation compound of formula (D).

[0082]

[0083] The divalent metal cation compound obtained in Example 1 was tested for specific binding performance with phosphatidylserine, and the specific test process and evaluation results are as follows.

[0084] The binding constant of the divalent metal cation compound and phosphatidylserine was measured by isothermal titration calorimetry (ITC) experiment: the divalent metal cation compound was dissolved in a mixed solution of ethanol / water with a volume ratio of 4 / 1 to 1.5 mM, and 0.08 mL was taken and added to the glass titration needle of the ITC measuring instrument. Then PC or PS was dissolved in a mixed solution of ethanol / water with a volume ratio of 4 / 1 to 0.15 mM, and 2.5 mL was taken and added to the reaction cell of the ITC measuring instrument. The titration number was set to 20, the first injection volume was 1 μL, and the subsequent 19 injection volumes were 2 μL each time. The measured titration curve was subtracted from the dilution heat to obtain the net reaction heat. The results were analyzed using a single binding site model to calculate the binding constant (Ka) and standard molar reaction enthalpy (ΔH).

[0085] Figure 3 The specific binding of the divalent metal cation compound to phosphatidylserine in Example 1 was evaluated as shown in the schematic diagram, Figure 3 where PS represents phosphatidylserine and PC represents phosphatidylcholine, as shown in Figure 3 Each peak represents the enthalpy change caused by the phosphatide-divalent metal cation compound, which is observed when the interaction between the phosphatide and the divalent metal cation compound is titrated. The heat of interaction of the divalent copper or zinc metal cation compound with phosphatidylserine (PS) is higher than that with phosphatidylcholine (PC), indicating that the affinity of the divalent copper or zinc metal cation compound for PS is significantly higher than that for PC. Through PEAQ-ITC analysis software calculation, the binding constant (Ka) of the divalent metal cation compound to PS is in the order of Zn < Cu < Ni.

[0086] According to Figure 3 The binding constant of the divalent metal cation compound to PS or PC is shown. Among the various divalent metal ions (Cu, Zn and Ni), copper ion is the best metal ion that can achieve specific binding to PS, and its binding constant is 1.76 x 10 5 mol -1 , indicating that the divalent copper metal cation compound in formula D is more conducive to specific binding to phosphatidylserine.

[0087] Test of the effect of the divalent metal cation compound obtained in Example 1 on the structure of the plasma membrane of cancer cells. The specific test process is as follows and the evaluation results are as follows:

[0088] 4T1 cells (1 x 10 5Example 1: Effect of bivalent copper metal cation compound on the membrane structure of cancer cells

[0089] Figure 4 The scanning electron microscope images of cancer cells treated with bivalent copper metal cation compound in Example 1 are schematically shown. The cancer cells treated with bivalent metal cation compound of Formula (D) were observed by scanning electron microscope, as shown in Figure 4 the electron micrograph showed that the plasma membrane of the cells was disturbed and the formation of pores on the cell membrane surface occurred in the field of view, indicating that the bivalent copper metal cation compound of Formula (D) could disturb the plasma membrane structure of cancer cells.

[0090] The test for stimulating cancer cells to release immunogenic substances by the bivalent copper metal cation compound obtained in Example 1 was carried out, and the specific test process and evaluation results are as follows.

[0091] The tumor cells in the logarithmic growth phase were trypsinized and prepared into a cell suspension, 5 x 10 4 cells were added to each well of a 24-well plate, and incubated for 12 h to allow the cells to adhere. Next, the cells were treated with CPT (7 μM) or bivalent copper metal cation compound (10, 20 or 30 μM) alone or in combination for 6 h. Subsequently, the old culture medium was removed and fresh culture medium was added, and incubation was continued for 18 h. The ATP concentration in the cell supernatant was measured using an ATP detection kit according to the manufacturer's instructions. After the specified treatment, the concentration of high mobility group box 1 (HMGB1) in the cell supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) kit. The luminescence value of ATP and the absorbance of HMGB1 were measured using a microplate reader (Thermo Scientific Varioskan Flash).

[0092] Figure 5 The release effect of immunogenic substances of cancer cells treated with bivalent metal cation compound in Example 1 of the present disclosure is schematically shown, as shown in Figure 5a-c shows that the extracellular ATP content of CT26 cells pretreated with divalent copper metal cation compounds increased significantly compared with the chemotherapy drug group. At a concentration of 30 μM, the extracellular ATP secretion of the experimental group pretreated with divalent copper metal cation compounds combined with CPT was 5.38 times higher than that of the control group. Similar results were also detected in B16F10 and 4T1 cells. As shown in Figure 5 d-f shows that the divalent copper metal cation compound experimental group has a higher level of HMGB1 release compared with the CT26 cells pretreated with chemotherapy drugs alone. Similar results were also detected in B16F10 and 4T1 cells. It is shown that the membrane disturbance of cancer cells induced by divalent copper metal cation compounds can effectively release immunogenic substances.

[0093] In vitro anti-tumor tests were performed on the divalent copper metal cation compounds obtained in Example 1, and the specific test process and evaluation results are as follows.

[0094] CT26, A549, B16F10, 4T1, L929, 3T3, MCF-10 and LO2 cells in the logarithmic growth phase were trypsinized and prepared into cell suspensions, 8000 cells were added to each well of a 96-well plate, and incubated for 12 h to allow the cells to adhere. The divalent copper metal cation compound was diluted with culture medium to a series of concentrations, the old culture medium was replaced, and after incubation for 24 h, the drug-containing culture medium was removed, 100 μL of 3-(4,5)-dimethylthiazol-2)-2,5-diphenyl tetrazolium bromide (MTT) solution (0.5 mg / mL) was added to each well, and incubated for another 4 h. Then the culture medium was discarded, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, the well plate was shaken to fully dissolve the purple formazan crystals, and the absorbance at 490 nm was measured using an enzyme label instrument (Thermo Scientific Varioskan Flash). The concentration range of the divalent copper metal cation compound was 20-150 μM, three parallel samples were set for each experimental concentration, and three independent experiments were performed.

[0095] Figure 6 The in vitro anti-tumor effect of the divalent copper metal cation compound in Example 1 of the present disclosure is schematically shown. As shown in Figure 6 The metal cation compound can achieve excellent in vitro anti-tumor effect in various cancer cells, while having less cytotoxicity to normal cells such as L929, 3T3, MCF-10 and LO2, indicating that the divalent metal cation compound has good cancer cell selectivity.

[0096] The in vitro PD-L1 expression reduction effect of the divalent metal cation compound obtained in Example 1 was tested, and the specific test process and evaluation results are as follows.

[0097] The effect of the metal cation compound on reducing the expression of PD-L1 in vitro was verified by Western blotting experiment analysis of the expression of specific proteins:

[0098] First, the CT26 cells or 4T1 cells were pretreated with the chemotherapeutic drug camptothecin (CPT, 7 μM) for 24 hours to further stimulate the phosphatidylserine everted (PS + cells) of the cancer cells, and then incubated in the medium containing different concentrations of divalent metal cation compounds to combine with the PS + cells. The CT26 cells or 4T1 cells were seeded in a 6-well plate at 10 6 cells per well, starved in the medium containing 250 nmol / L Gas6 for 6 hours, and then co-cultured with PS + cells (5 x 10 6 cells / well).

[0099] After 12 hours, the PS + cells were washed away with PBS, and the CT26 cells or 4T1 cells were continued to be cultured in the medium containing 0.5% FBS for 12 hours. Then, the total protein of the cells was extracted and subjected to Western blotting analysis. Next, the medium was removed, the cells were lysed by adding 200 μL RIPA lysis buffer combined with 1% PMSF (100 mM) and 2% phosphatase inhibitor, pipetted and the sample was collected.

[0100] The cell sample was centrifuged at a speed of 12000 rpm for 15 minutes at 4°C, and then the supernatant was collected. The protein concentration was determined using a BCA protein assay kit. The appropriate amount of protein was resuspended in the loading buffer, heated at a temperature of 100°C for 10 minutes, then separated on 8% SDS-PAGE, and transferred to a 0.45 μm PVDF membrane in the transfer buffer.

[0101] After incubation in the blocking buffer for 1 hour to saturate the non-specific binding sites, the primary antibodies were incubated at a temperature of 4°C overnight, including anti-PD-L1 (1:5000), anti-AKT (1:5000), anti-p-AKT (1:5000) and anti-β-actin (1:2000).

[0102] The PVDF membrane was washed with TBST three times, and then incubated with the secondary antibody at a temperature of 20-35°C for 1 hour, followed by washing and chemiluminescence detection, and then imaged using ImageQuant LAS4000.

[0103] Figure 7The in vitro evaluation of the bivalent metal cation compound in Example 1 of the present disclosure to reduce PD-L1 expression is schematically shown as follows: Figure 7 As can be seen from the experimental results, the bivalent metal cation compound can reverse the increase in PD-L1 expression, and even reduce the expression level of PD-L1 to a level lower than that of the control group. At the same time, the expression of p-AKT is down-regulated, while the total AKT protein level does not change. Therefore, the cancer cell surface phosphatidylserine evagination induced by the chemotherapeutic drug can activate and enhance the PS-AKT-PD-L1 axis, and the destruction of this signal axis by the bivalent metal cation compound can lead to a decrease in PD-L1 expression.

[0104] The in vivo anti-tumor immunotherapy of the bivalent metal cation compound obtained in Example 1 was tested, and the specific testing process and evaluation results are as follows.

[0105] Among them, all animal experiments are carried out in accordance with the guidelines of the Laboratory Animal Guidelines. In the therapeutic CT26 tumor model, CT26 cells are inoculated into BALB / c mice.

[0106] When the CT26 tumor volume approaches 80mm 3 , the mice are divided into 4 groups, namely PBS group (1), CPT (3mg / kg) group (2), CPT (3mg / kg) + aPD-L1 (5mg / kg) group (3) and CPT (3mg / kg) + bivalent metal cation compound (10mg / kg) group (4). Inject CPT into the tail vein of the mouse, and 2 hours after intravenous injection, inject aPD-L1 or bivalent metal cation compound into the tail, once every 1 day, a total of three treatments. Measure the tumor volume and weight every 2 days to evaluate the anti-tumor therapeutic effect of the metal cation compound.

[0107] The calculation formula of tumor volume (V) is V = 0.5 x L x W 2 , where L and W represent length and width, respectively. After 3 cycles of treatment, the tumor and lymph node are collected on day 5. T lymphocytes are separated using lymphocyte separation medium and stained with FITC anti-mouse CD3, APC anti-mouse CD4 and PerCP / Cyanine5.5 anti-mouse CD8a antibodies, and then the intratumoral infiltration of T lymphocytes is detected by flow cytometry.

[0108] The tumor-draining lymph node is stained with PerCP / Cyanine5.5 anti-CD11c, FITC anti-CD80 and APC anti-CD86 antibodies, and then the maturity of DC in the tumor-draining lymph node is detected by flow cytometry.

[0109] Figure 8 Fig. 1 shows the in vivo anti-tumor immunotherapy effect of the divalent metal cation compound in Example 1. As shown in Fig. 1, the tumor volume, mouse weight and survival rate of the four groups of mice after intravenous injection are shown, wherein the divalent metal cation compound treatment leads to rapid and stable tumor regression, and the survival rate of the mice is as high as 70%, showing a good in vivo anti-tumor effect. Figure 8 Figure 8 a-c show the changes in tumor volume, mouse weight and survival rate after intravenous injection of the four groups of mice, wherein the divalent metal cation compound treatment leads to rapid and stable tumor regression, and the survival rate of the mice is as high as 70%, showing a good in vivo anti-tumor effect. Figure 8 d-i show the in vivo anti-tumor immunotherapy effect of the divalent metal cation compound, and as can be seen from the figure, the divalent metal cation compound can effectively induce DC maturation and further enhance the expression of MHC II, which is helpful for antigen presentation. After intravenous injection treatment, the CD4 + , CD8 + T cells in the tumor are significantly more than those in other groups, indicating that the metal cation compound promotes the enrichment of T cells in tumor cells.

[0110] Example 2

[0111] A method for preparing a divalent metal cation compound, comprising the following steps:

[0112] (1) In a 100 mL flask, add terpyridine (100 mg, 0.36 mmol), dicyclohexyl carbodiimide (DCC) (112 mg, 0.54 mmol) and 4-dimethylaminopyridine (DMAP) (13 mg, 0.11 mmol) and dissolve in 5 mL of DMF solution. Dissolve hexaethylene glycol (509 mg, 1.81 mmol) in DMF.

[0113] (2) Add the hexaethylene glycol solution to the 5 mL DMF reaction solution using a constant pressure dropping funnel within 30 min, and react at 20°C overnight. After stirring overnight, cool the mixture and remove the solvent under vacuum. Purify the crude product by column chromatography (dichloromethane:methanol = 15:1) to obtain the metal cation compound precursor as a white solid. Figure 9 Fig. 2 shows the nuclear magnetic resonance hydrogen spectrum of the metal cation compound precursor in Example 2, as shown in Fig. 2, the molecular formula of the compound precursor can be obtained. Figure 9

[0114] (3) Dissolve 0.21 mmol of the metal cation compound precursor in methanol, and add a methanol solution containing zinc nitrate (0.21 mmol). Ultrasonically treat the mixed solution at 20-35°C for 60 min, then remove the solvent under vacuum, and finally dry to obtain the divalent metal cation compound of formula (E).

[0115] ​​

[0116] The specific binding performance of the divalent metal cation compound obtained in Example 2 with phosphatidylserine was tested. The specific test process and evaluation results are as follows.

[0117] The binding interaction between divalent metal cations and phosphatidylserine was measured using flow cytometry: 4T1 cells in logarithmic growth phase were digested with trypsin and a cell suspension was prepared. 1×10⁻⁶ cells were added to each well of a 12-well plate. 5 After incubating 4T1 cells for 12 hours to allow them to adhere, the cells were pretreated with 7.0 μM camptothecin (CPT) for 24 hours to induce greater phosphatidylserine exposure on the tumor cell surface, and then PS were obtained. + Cells. Collect PS + Cells were co-incubated with different concentrations of divalent metal cation compounds to bind CPT-induced PS. + Phosphatidylserine on the cell surface. Subsequently, Annexin V-FITC, a FITC-labeled annexin from an apoptosis detection kit, was used to specifically bind to phosphatidylserine. After staining, the fluorescence intensity of FITC-positive cells in each group was analyzed by flow cytometry (FCM) in the FITC channel to detect the amount of phosphatidylserine bound.

[0118] Figure 10 This diagram schematically illustrates the evaluation of the specific binding of the divalent metal cation compound to phosphatidylserine in Example 2 of this work. It should be noted that PS in the diagram represents phosphatidylserine. Figure 10 As shown, induction with the chemotherapy drug camptothecin can promote the outward turning of more phosphatidylserine from the inner lobes, resulting in cancer cells with a stronger PS positive signal (PS). + (cells). Subsequently, co-incubation of PS-positive cells with different concentrations of divalent metal cation compounds reduced PS exposure to varying degrees and allowed PS to bind to the divalent metal cation compounds. Once bound, PS could no longer bind to FITC-labeled annexin V, leading to a decrease in Annexin V fluorescence. This indicates that the divalent metal cation compound of formula (E) can not only specifically bind to PS, but also shield PS from interactions with other proteins or receptors.

[0119] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of this disclosure. It should be understood that the above descriptions are merely specific embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. Use of a divalent metal cation compound for the preparation of a medicament for immunomodulation and antitumor by binding to phosphatidylserine on the surface of tumor cells, wherein, The divalent metal cation compound has a structural formula shown in formula (I): Formula (I) wherein M is selected from any one of Cu 2+ , Zn 2+ , Ni 2+ ; R1is selected from a single bond; X is selected from O; R2is selected from or .

2. The use according to claim 1, wherein, The binding affinity constant of the divalent metal cation compound to phosphoserine is 2.41-10.6 x 10 -6 M.

3. The use according to claim 1, wherein, The divalent metal cation compound is suitable for inhibiting the activation of downstream AKT signaling pathway to inhibit the expression of PD-L1.

4. A preparation method of a divalent metal cation compound used in the application of any one of claims 1-3, comprising the following steps: reacting compound (A) and compound (B) in an organic solvent to obtain compound (C); Formula (A) Formula (B) reacting the compound (C) with a divalent metal salt in an organic solvent to obtain the compound of formula (I); Formula (C).

5. The production method according to claim 4, wherein The divalent metal salt includes at least one of nickel salt, copper salt, zinc salt.

6. The production method according to claim 4, wherein The divalent metal salt is zinc nitrate or copper nitrate.

7. The production method according to claim 4, wherein The reaction of the compound (A) and the compound (B) in the organic solvent includes: reacting the compound (A) and the compound (B) in the presence of dicyclohexyl carbodiimide and 4-dimethylaminopyridine under an inert gas atmosphere.

8. The production method according to claim 7, wherein The reaction temperature is 20-50℃, and the reaction time is 2 hours to 24 hours; The reaction temperature of the compound (C) and the divalent metal salt is 20-40℃, and the reaction time is 1 hour to 24 hours.

9. A divalent metal cation compound suitable for the application of any one of claims 1-3, the divalent metal cation compound has a structural formula shown in formula (I): Formula (I) wherein, M is selected from any one of Cu 2+ , Zn 2+ , Ni 2+ ; R1is selected from a single bond; X is selected from O; R2is selected from or .

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