CN118812426B - Divalent metal cation compounds, their preparation methods and applications - Google Patents
Divalent metal cation compounds, their preparation methods and applicationsInfo
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Abstract
本公开提供了一种二价金属阳离子化合物及其制备方法和应用，属于医药治疗领域。该化合物具有式(I)所示的结构式，其中，R1选自单键、取代或非取代的C1‑5链式亚烃基、取代或未取代的C6‑12亚芳基；X选自O、S和NH；R2选自C1‑30疏水链烃基或聚醚，其中聚醚的数均分子量为200‑2500。本公开的二价金属阳离子化合物能够与肿瘤细胞表面的磷酯酰丝氨酸结合，促进树突细胞成熟并抑制PD‑L1表达，可应用于制备免疫调节和抗肿瘤的药物中。This disclosure provides a divalent metal cation compound, its preparation method, and its application, belonging to the field of pharmaceutical therapy. The compound has the structural formula shown in formula (I). Wherein, R1 is selected from single bonds, substituted or unsubstituted C1-5 chain alkylene groups, substituted or unsubstituted C6-12 arylene groups; X is selected from O, S, and NH; R2 is selected from C1-30 hydrophobic chain hydrocarbon groups or polyethers, wherein the number average molecular weight of the polyether is 200-2500. The divalent metal cation compound disclosed herein can bind to phosphatidylserine on the surface of tumor cells, promoting dendritic cell maturation and inhibiting PD-L1 expression, and can be used in the preparation of immunomodulatory and antitumor drugs.
Description
Technical Field
The present disclosure relates to the field of medical treatment, and in particular to a divalent metal cation compound, a preparation method thereof, and an application thereof in a medicament for inhibiting PD-L1 and realizing an immune anti-tumor effect.
Background
Cancer is a major cause of death in the global population, which severely hampers the prolongation of life expectancy in humans. Checkpoint blocking therapy against anti-apoptosis-ligand 1 (PD-L1)/apoptosis-1 (PD-1) can facilitate T cell-mediated immune monitoring against tumor cells, significantly altering the treatment prospects of advanced cancers.
In the related art, the main problems of immune checkpoint therapy are low response rate, insensitivity of many patients to immune checkpoint therapy, lack of enough tumor specific antigen, lack of tumor-reactive T cells, up-regulation of PD-L1 expression after some patients receive immune checkpoint therapy, treatment resistance, reduced treatment effect, and risk of metastasis and recurrence, and adverse reactions caused by combination with PD-L1 on the surface of normal cells, resulting in toxic and side effects. To solve the problems, a novel and efficient cancer cell targeting mechanism needs to be developed, the toxic and side effects on normal tissues are reduced, the mechanism for deeply reducing PD-L1 expression is developed, and a high-efficiency anti-tumor immunity medicament is constructed. The research has very good academic research value and also has very good social requirements.
Disclosure of Invention
In view of the above, the present disclosure proposes to construct an organometallic complex having specific binding interactions with phosphatidylserine on the surface of tumor cell membranes, which can selectively bind with phosphatidylserine on tumor cell membranes to achieve accurate targeting of tumors, and secondly, induce cancer cells to release a large amount of immunogenic substances by disturbing the plasma membrane structure, promote DC maturation and antigen presentation, improve the tumor ' desert ' -type ' immunosuppressive microenvironment, enhance the infiltration and activity of T cells, and finally, shield phosphatidylserine signals to block interactions with receptors, inhibit activation of downstream AKT signaling pathway, down regulate PD-L1 expression deeply, not generate drug resistance compared with conventional PD-L1 antibodies, not rely on negative feedback mechanisms of antibody reversal tumor immunity, and reactivate T cell recognition, thereby effectively killing tumor cells.
It is an object of the present disclosure to provide a divalent metal cation compound.
Another object of the present disclosure is to provide a method for preparing the above divalent metal cation compound.
It is a further object of the present disclosure to provide the use of the divalent metal cation compound described above.
The above object of the present disclosure is achieved by the following means.
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, R 1 is selected from single bond, substituted or unsubstituted C 1-5 chain hydrocarbylene, substituted or unsubstituted C 6-12 arylene, X is selected from O, S and NH, R 2 is selected from C 1-30 hydrophobic chain hydrocarbylene or polyether, wherein the polyether has a number average molecular weight of 200-2500.
In some embodiments, the divalent transition metal M comprises Ni, cu, zn, cd, pb, co, fe, ir or Ti.
In some embodiments, R 1 is selected from a single bond, a substituted or unsubstituted C 1-5 chain alkylene, a substituted or unsubstituted C 6-12 arylene, preferably R 1 is a single bond or a C 1-5 chain alkylene.
In some embodiments, R 1 is selected from one of the following structures:
In some embodiments, R 2 is selected from a C 1-16 chain alkyl or a polyethylene glycol chain, such as a C 1-8 chain alkyl.
In some embodiments, R 2 is selected from C 1-30 alkyl or C 2-30 alkenyl, e.g., C 1-20 alkyl or C 2-20 alkenyl, e.g., C 1-16 alkyl or C 2-16 alkenyl, e.g., C 1-8 alkyl or C 2-8 alkenyl.
In some embodiments, R 2 is selected from:
In some embodiments, the divalent metal cation compound has an affinity for phosphatidylserine on the surface of cancer cells, a binding affinity of 2.41-10.6X10 -6 M, and binding energy effective to mask phosphatidylserine signals.
According to an embodiment of another aspect of the present disclosure, there is provided a method for preparing a divalent metal cation compound having immunomodulatory and antitumor effects represented by formula I, comprising:
(1) Reacting the compound (A) with the compound (B) in an organic solvent to obtain a compound (C);
(2) Reacting the compound (C) with a divalent metal salt in an organic solvent to obtain the divalent metal cation compound of the above formula I.
In some embodiments, the hydrophobic or polyether structure of formula R 2 -X-H includes the following structures:
in some embodiments, the divalent metal salt comprises 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.
In some embodiments, the reaction in step (1) is performed in a solution of Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP). In some embodiments, the reaction temperature in step (1) is 20-50 ℃ and the reaction time is 2 hours to 24 hours.
In some embodiments, the reaction in step (1) is further preferably carried out in the presence of Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) and in an inert atmosphere, the reaction temperature in step (1) being 20-50 ℃ and the reaction time being 4-12 hours.
In some embodiments, the reaction temperature in step (2) is 20-40 ℃ and the reaction time is 1 to 24 hours.
According to an embodiment of another aspect of the present disclosure, there is provided the use of the divalent metal cation compound described above for the preparation of a medicament for immunomodulation and anti-tumour.
In some embodiments, the metal cation compounds described above are useful as immunomodulating and antitumor agents, capable of targeting phosphatidylserine on the surface of cancer cells, and have cancer cell selectivity. The compound has the effects of inhibiting tumor cells in vitro and in vivo, has an IC50 of 18.65-37.92 mu M for a cancer cell line, and has the function of activating immune cells through in vitro and in vivo experiments. The medicine can block the interaction between phosphatidylserine and phosphatidylserine receptor, inhibit the activation of downstream AKT signal path and inhibit the expression of PD-L1, and has the functions of inhibiting the expression of PD-L1 and playing the role of immunoregulation and anti-tumor.
Based on the technical scheme, the divalent metal cation compound provided by the disclosure and the preparation method and application thereof have one or a part of the following beneficial effects:
Phosphatidylserine is an important component of human cells and is usually present inside the cell membrane. However, in cases of tumor microenvironments such as hypoxia, low pH and disturbed calcium homeostasis, the exposure of phosphatidylserine to the extracellular leaves of cancer cells is further enhanced. The exogenized phosphatidylserine not only alters the biochemical and physical properties of the plasma membrane, but also initiates a series of interactions between endogenous extracellular proteins and receptors on neighboring cells, creating an immunosuppressive microenvironment and promoting tumor growth and metastasis. The divalent metal cation compound disclosed by the disclosure can perform strong coordination with phosphatidylserine on the surface of cancer cells, and phosphatidylserine is hardly present on the outer membrane of normal cells, so that the metal cation compound can effectively target tumors. Because the tumor antigen and the adjuvant are released by disturbing the plasma membrane structure of cancer cells, the dendritic cells are stimulated to mature, and the tumor immunosuppression microenvironment is improved. After the interaction of phosphatidylserine and a receptor thereof is blocked, the downstream AKT signal phosphorylation is further blocked, the PD-L1 expression is reduced, the immune negative feedback mechanism is reversed, the recognition and elimination of cytotoxic T lymphocytes on cancer cells are activated, and the tumor immunotherapy is effectively realized.
The metal cation compound can target tumors and reduce toxic and side effects on normal tissues. The metal cation compound has simple structure and great potential for clinical transformation. Compared with the traditional immune checkpoint antibody drug, the novel anti-tumor immune drug directly reduces PD-L1 expression from the source, does not generate antibody treatment drug resistance, and can inhibit tumor metastasis.
Drawings
The present disclosure is described in further detail below with reference to the accompanying drawings.
FIG. 1 schematically illustrates a schematic synthesis of a divalent metal cation compound according to an embodiment of the present disclosure;
FIG. 2 schematically shows a nuclear magnetic resonance hydrogen spectrum of a divalent metal cation compound precursor in example 1 of the present disclosure;
Fig. 3 schematically shows an evaluation schematic of specific binding of a divalent metal cation compound to phosphatidylserine (abbreviated as PS) in example 1 of the present disclosure, wherein a to c are time-dependent heat absorption curves of a divalent copper metal ion compound, a divalent zinc metal ion compound, and a divalent nickel metal ion compound to PS/PC, and d to f are time-dependent enthalpy values of the divalent copper metal ion compound, the divalent zinc metal ion compound, and the divalent nickel metal ion compound to PS/PC;
FIG. 4 schematically shows a Scanning Electron Microscope (SEM) image of cancer cells after treatment with a divalent metal cation compound according to example 1 of the present disclosure, wherein a is an SEM image of a control group of cancer cells, b is an enlarged image of a, c is an SEM image of a 30 μm concentration group of cancer cells, d is an enlarged image of c, e is an SEM image of a 60 μm concentration group of cancer cells, and f is an enlarged image of e;
fig. 5 schematically shows a graph of the effect of the release of an immunogenic substance from cancer cells after treatment with a divalent metal cation compound according to example 1 of the present disclosure, wherein a to c are the results of cellular ATP changes for cancer cells CT26, B16F10 and 4T1, respectively, and d to F are the results of fold change in extracellular HMGB1 release from cancer cells CT26, B16F10 and 4T1, respectively;
FIG. 6 schematically shows an in vitro anti-tumor effect graph of divalent metal cation compounds of example 1 of the present disclosure, wherein a to d are cytotoxicity results of cancer cells CT26, A549, B16F10, and 4T1, respectively, and e to h are cytotoxicity results of normal cells L929, 3T3, MCF-10, and LO2, respectively;
FIG. 7 schematically shows a schematic of evaluation of in vitro reduction of PD-L1 expression by divalent metal cation compounds according to example 1 of the present disclosure, wherein a is the result of Western blotting of PD-L1 protein in CT26 cells and b is the result of Western blotting of PD-L1 protein in 4T1 cells;
FIG. 8 schematically shows an in vivo anti-tumor immunotherapy effect graph of the divalent metal cation compound of example 1 of the present disclosure, wherein a is a change curve of tumor volume of mice, b is a change curve of body weight of mice, c is a change curve of survival rate of mice, d is a flow cytometry analysis histogram of mature dendritic cells, e and f are respectively statistics of the ratio of mature dendritic cells in total dendritic cells, g is a T cell immunohistochemical staining chart, h is statistics of the ratio of CD8 + T cells in total T cells, i is statistics of the ratio of CD4 + T cells in total T cells;
FIG. 9 schematically shows a nuclear magnetic resonance hydrogen spectrum of a metal cation compound precursor in example 2 of the present disclosure, and
FIG. 10 schematically shows an evaluation of specific binding between a divalent metal cation compound and phosphatidylserine in example 2.
Detailed Description
The externalized phosphatidylserine not only alters the biochemical and physical properties of the plasma membrane, but also initiates interactions between endogenous extracellular proteins and receptors on neighboring cells, forming an immunosuppressive microenvironment, and promoting tumor growth and metastasis. In the process of realizing the present disclosure, it was found that a hydrophilic portion formed by coordination of terpyridine and divalent transition metal ions can specifically bind to phosphatidylserine on the surface of cancer cells, thereby realizing a targeting effect on tumors.
In view of this, the present disclosure constructs an organometallic complex having a specific binding interaction with phosphatidylserine on the surface of a tumor cell membrane, comprising the above-described hydrophilic portion and a hydrophobic segment or a hydrophilic segment linked to the hydrophilic portion. Through the interaction of the dendritic cell and the phosphatidylserine, the maturation of the dendritic cell can be promoted, and the expression of PD-L1 can be reduced, so that the immunotherapy of tumors can be realized.
For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same.
In an embodiment of the present disclosure, there is provided a divalent metal cation compound of formula I, M is selected from divalent transition metal elements, R 1 is selected from single bond, substituted or unsubstituted C 1-5 chain hydrocarbylene, substituted or unsubstituted C 6-12 arylene, X is selected from O, S and NH, R 2 is selected from C 1-30 hydrophobic chain hydrocarbylene or polyether, wherein the polyether has a number average molecular weight of 200-2500.
In the embodiment of the disclosure, the divalent metal cation compound shown as the formula 1 consists of a hydrophilic head part and a hydrophilic or hydrophobic tail part, wherein the hydrophilic head part is formed by coordination of a terpyridine part and divalent transition metal ions, can be well coordinated and combined with phosphatidylserine, so that plasma membrane structure is disturbed, cancer cells are induced to activate and release a large amount of immunogenic substances, further mask phosphatidylserine signals, down regulate the expression of PD-L1, and the hydrophilic or hydrophobic tail part is formed by hydrophobic chain alkyl or polyether, plays a role in masking the phosphatidylserine, blocks interaction between the phosphatidylserine and a receptor and inhibits downstream AKT signal path activation.
In embodiments of the present disclosure, R 2 is selected from C 1-16 chain hydrocarbyl or polyethylene glycol chain, preferably R 2 is selected from C 1-8 chain hydrocarbyl.
In embodiments of the present disclosure, R 2 is selected from C 1-30 alkyl or C 2-30 alkenyl, preferably R 2 is selected from C 1-20 alkyl or C 2-20 alkenyl, preferably R 2 is selected from C 1-16 alkyl or C 2-16 alkenyl, preferably R 2 is selected from C 1-8 alkyl or C 2-8 alkenyl, more preferably R 2 is selected from one of the following structures:
In the embodiment of the disclosure, when R 2 is a hydrophobic chain such as alkyl or alkenyl, interaction between phosphoserine and a phosphoserine receptor can be blocked, signals of the phosphoserine are shielded, activation of a downstream AKT signal pathway is inhibited, and thus PD-L1 expression is inhibited, and when R 2 is a polyethylene glycol chain, the effect of shielding signals can be further enhanced.
In an embodiment of the disclosure, R 1 is selected from a single bond, phenylene, or biphenylene. During the screening of R 1, R 1 is selected from one of the following structures, so that the divalent metal cation compound has more stable structure and better coordination with phosphatidylserine:
In embodiments of the present disclosure, M is selected from Ni, cu, zn, cd, pb, co, fe, ir or Ti, more preferably Cu, and the divalent metal cation compound has an affinity for phosphoserine on the surface of cancer cells, and the binding affinity constant of the two is 2.41-10.6X10 -6 M, and the binding of the two can effectively shield the interaction of phosphoserine and a phosphoserine receptor.
There is also provided, in accordance with an embodiment of the present disclosure, a method of preparing a divalent metal cation compound having immunomodulatory and antitumor effects represented by formula I, comprising:
(1) Reacting the compound (A) with the compound (B) in an organic solvent to obtain a compound (C);
(2) Reacting the compound (C) with a divalent metal salt in an organic solvent to obtain the divalent metal cation compound of the above formula I.
According to the embodiment of the disclosure, a hydrophilic head part is formed by coordination of a terpyridine ring and divalent transition metal ions, and a hydrophilic or hydrophobic tail part is connected with the hydrophilic head part through condensation reaction. Fig. 1 schematically shows a schematic synthesis of a divalent metal cation compound according to an embodiment of the present disclosure. In some embodiments, the synthesis of a particular metal cationic compound of formula I is shown in fig. 1.
In some embodiments of the present disclosure, the reaction in step (1) is performed in Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) solutions. The reaction temperature in step (1) is 20-50 ℃, e.g. 30 ℃, 35 ℃, 40 ℃ and 45 ℃, and the reaction time is 2-24 hours, e.g. 4-8-12-16-20 hours.
In some embodiments of the present disclosure, the reaction in step (1) is further preferably performed in the presence of Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) and in an inert atmosphere, and the reaction temperature in step (1) is 20-50 ℃ and the reaction time is 2 to 24 hours.
In some embodiments of the present disclosure, the reaction temperature in step (2) is 20-40 ℃, e.g., 25 ℃, 30 ℃, 35 ℃, and the reaction time is 1 hour to 24 hours, e.g., 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours.
According to an embodiment of the present disclosure, the organic solvent in steps (1) and (2) is each independently selected from chloroform, dichloromethane, ethyl acetate, methanol, ethanol, toluene, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N-dimethylformamide, and any combination thereof.
In some embodiments of the present disclosure, 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, 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.
According to an embodiment of the present disclosure, there is also provided an application of the divalent metal cation compound in preparing a medicament for immunomodulation and anti-tumor.
In some embodiments of the present disclosure, divalent metal cation compounds can target tumors, reducing toxic side effects on normal tissues. The metal cation compound has simple structure and great potential for clinical transformation.
According to embodiments of the present disclosure, the divalent metal cation compounds provided herein specifically bind to phosphatidylserine on the surface of tumor cells and are useful for inhibiting activation of downstream AKT signaling pathways to inhibit PD-L1 expression.
In some embodiments of the present disclosure, compared to traditional immune checkpoint antibody drugs, the novel anti-tumor immune drug can directly reduce PD-L1 expression from the source, does not produce antibody treatment resistance, and can inhibit tumor metastasis, effectively realizing tumor immunotherapy.
The present disclosure is further illustrated by the following examples. In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough explanation of the disclosed embodiments. It may be evident, however, that one or more embodiments may be practiced without these specific details. Moreover, the details of the various embodiments below may be arbitrarily combined into other viable embodiments without conflict.
It should be noted that the following specific examples are given by way of illustration only and the scope 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 the Living technologies Co., gmbH, wuhanposai, china. Methods such as immunofluorescent staining are well known in the art and may be performed by textbooks or descriptions of related documents, and are not described in detail.
Example 1
A process for the preparation of a divalent metal cation compound comprising the steps of:
(1) To a 100mL flask were added terpyridine (100 mg,0.36 mmol), dicyclohexylcarbodiimide (DCC) (112 mg,0.54 mmol), and 4-Dimethylaminopyridine (DMAP) (13 mg,0.11 mmol), and dissolved in 5mL of DMF. Farnesol (53 mg,0.24 mmol) was dissolved in DMF.
(2) The farnesol solution was added to 5mL of DMF solution over 30min with a constant pressure dropping funnel and reacted overnight at 20 ℃. After stirring overnight, the mixture was cooled and the solvent was removed under vacuum. The crude product was purified by column chromatography (dichloromethane: methanol=15:1) to give the metal cation compound precursor as a white solid. Fig. 2 schematically shows a nuclear magnetic resonance hydrogen spectrum of a metal cation compound precursor in this example 1, and the molecular formula of the compound precursor can be obtained as shown in fig. 2.
(3) 0.21Mmol of the metal cation compound precursor was dissolved in a methanol solution, and a methanol solution in which copper nitrate (0.21 mmol), zinc nitrate (0.21 mmol) or nickel nitrate (0.21 mmol) was dissolved was added, respectively. The mixed solution is sonicated at 20-35 ℃ for 60min, followed by removal of the solvent under vacuum, and finally dried to give the divalent metal cation compound of formula (D).
The divalent metal cation compound obtained in example 1 was tested for specific binding properties to phosphatidylserine, and specific test procedures and evaluation results are as follows.
The binding constants of the divalent metal cation compound and phosphatidylserine were measured using an isothermal titration quantitative thermal (ITC) experiment by dissolving the divalent metal cation compound to 1.5mM with a mixed solution of ethanol/water at a volume ratio of 4/1, and adding 0.08mL to a glass titration needle of an ITC meter. Then, PC or PS was dissolved in a mixed solution of ethanol/water volume ratio of 4/1 to 0.15mM, and 2.5mL was added to the reaction cell of the ITC-measuring apparatus. The number of titrations was set to 20, the first injection volume was 1 μl and the subsequent 19 injections volumes were 2 μl each. The heat of dilution is subtracted from the measured titration curve to give the net heat of reaction. The results were analyzed using a single binding site model, and binding constants (Ka) and standard molar reaction enthalpies (Δh) were calculated.
FIG. 3 schematically shows an evaluation of specific binding of the divalent metal cation compound to phosphatidylserine in the present example 1, in FIG. 3, PS represents phosphatidylserine, PC represents phosphatidylcholine, and each peak represents a change in enthalpy value caused by the phospholipid-divalent metal cation compound, as shown in FIG. 3, and it is observed that the interaction of the divalent copper or zinc metal cation compound with Phosphatidylserine (PS) is higher in heat absorption than the interaction of the divalent copper or zinc metal cation compound with Phosphatidylserine (PC), indicating that the affinity of the divalent copper or zinc metal cation compound to PS is significantly higher than that to PC. The order of binding constants (Ka) of divalent metal cation compounds to PS was Zn < Cu < Ni as calculated by PEAQ-ITC analysis software.
According to the binding constant of the divalent metal cation compound with PS or PC shown in fig. 3, among various divalent metal ions (Cu, zn and Ni), copper ion is the best metal ion for achieving PS-specific binding, and its binding constant is 1.76×10 5mol-1. The cupric metal cation compound in formula D is illustrated to be more favorable for specific binding to phosphatidylserine.
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 procedure and evaluation result are as follows:
4T1 cells (1X 10 5/well) were seeded in 12-well plates with 13mm round glass cover plates at the bottom, incubated with varying concentrations of cupric metal ion compound (30 or 60. Mu.M) for 1h, the medium removed and rinsed three times with Phosphate Buffer (PBS), the cells fixed with 2.5% glutaraldehyde solution for 2h, and then dehydrated through a series of gradient increasing ethanol solutions (30%, 40%,50%,70%,90%, 100%) at room temperature for 10min. After drying overnight, the gold was evaporated and the tumor cells were observed by scanning electron microscopy (GEMINISEM, zeiss, germany).
Fig. 4 schematically shows a scanning electron microscope image of cancer cells treated with the cupric metal cation compound of this example 1. The scanning electron microscope was used to observe the cancer cells treated with the divalent metal cation compound of formula (D), and as shown in fig. 4, the electron microscope photograph showed that plasma membrane disruption of the cell membrane occurred, and that the occurrence of holes on the surface of the cell membrane occurred in the visual field, indicating that the divalent copper metal cation compound of formula (D) could disrupt the plasma membrane structure of the cancer cells.
The cupric metal cation compound obtained in example 1 was tested for the release of an immunogenic substance from cancer cells, and the specific test procedure and evaluation results were as follows.
Tumor cells in the logarithmic growth phase were digested with pancreatin and made into cell suspensions, 5×10 4 cells were added per well in 24-well plates, and incubated for 12h waiting for cell attachment. Next, the cells were treated with CPT (7. Mu.M) or cupric metal cation compound (10, 20 or 30. Mu.M), respectively, alone or in combination for 6h. Subsequently, the old medium was removed and new medium was added and incubation was continued for 18h. The ATP concentration of the cell supernatant was measured using an ATP assay kit according to the manufacturer's instructions. After the prescribed treatment, the concentration of high mobility group box B1 (HMGB 1) 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).
Fig. 5 schematically shows a graph of the effect of release of an immunogenic substance from cancer cells after treatment with a divalent metal cation compound according to example 1 of the present disclosure, as shown in fig. 5a-c, the CT26 extracellular ATP content of the divalent copper metal cation compound pretreatment is significantly increased compared to the chemotherapeutic drug group. At a concentration of 30. Mu.M, the extracellular ATP secretion of the experimental group pretreated with the cupric metal cation compound in combination 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 fig. 5d-f, the cupric metal cation compound experimental group had higher levels of HMGB1 release compared to CT26 cells pretreated with chemotherapeutic alone. Similar results were also detected in B16F10 and 4T1 cells. Indicating that the cancer cell membrane disturbance induced by the cupric metal cation compound can effectively release the immunogenic substances.
The cupric metal cation compound obtained in example 1 was subjected to in vitro antitumor test, and the specific test procedure and evaluation results are as follows.
CT26, A549, B16F10, 4T1, L929, 3T3, MCF-10 and LO2 cells in the logarithmic phase were digested with pancreatin and made into cell suspensions, 8000 cells were added to each well of a 96-well plate, and incubated for 12 hours waiting for cell attachment. The cupric metal cation compound was diluted with medium to a series of concentrations, the old medium was replaced, after incubation for 24 hours, the medicated medium was removed, and 100 μl of 3- (4, 5) -dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL) was added to each well for further incubation for 4 hours. The medium was then discarded, 150 μl of dimethyl sulfoxide (DMSO) was added to each well, the well plate was shaken to dissolve the purple crystalline formazan sufficiently, and the absorbance at 490nm was measured using an microplate reader (Thermo Scientific Varioskan Flash). The concentration range of the bivalent copper metal cation compound is 20-150 mu M, three parallel samples are arranged for each experiment concentration, and three independent experiments are carried out.
Fig. 6 schematically shows an in vitro antitumor effect graph of the cupric metal cation compound in example1 of the present disclosure. As shown in fig. 6, the metal cation compound can achieve excellent in vitro anti-tumor effect in various cancer cells, and has low cytotoxicity to normal cells such as L929, 3T3, MCF-10, LO2 and the like, which indicates that the divalent metal cation compound has good cancer cell selectivity.
The in vitro PD-L1 expression reducing effect of the divalent metal cation compound obtained in example 1 was tested, and the specific test procedure and evaluation results are as follows.
The expression of specific proteins is analyzed by Western blotting experiments to verify that the metal cation compound has the effect of reducing the expression of PD-L1 in vitro:
CT26 cells or 4T1 cells were first pre-treated with the chemotherapeutic drug camptothecine (CPT, 7 μm) for 24 hours to further stimulate cancer cell phosphatidylserine eversion (PS + cells) and then incubated in medium containing different concentrations of divalent metal cation compound to bind to PS + cells. CT26 cells or 4T1 cells were seeded at 10 6 cells per well in 6-well plates, starved cultured in medium containing 250nmol/L Gas6 for 6 hours, and co-cultured with PS + cells (5X 10 6 cells/well).
After 12 hours, PS + cells were washed away with PBS and CT26 cells or 4T1 cells were continuously cultured in medium containing 0.5% fbs for 12 hours. Then, the total cell proteins were extracted and subjected to Western blotting analysis. The medium was then removed, the cells were lysed by adding 200 μl of RIPA lysis buffer in combination with 1% pmsf (100 mM) and 2% phosphatase inhibitor, and the samples were pipetted and collected.
Cell samples were centrifuged at 12000rpm for 15 minutes at 4 ℃ and the supernatant was collected. Protein concentration was determined using BCA protein assay kit. Appropriate amounts of protein were resuspended in loading buffer, heated at 100℃for 10 min, then separated on 8% SDS-PAGE and transferred in transfer buffer to a 0.45 μm PVDF membrane.
The non-specific binding sites were saturated by incubation in blocking buffer for 1 hour and then incubated overnight at 4℃with several primary antibodies, anti-PD-L1 (1:5000), anti-AKT (1:5000), anti-p-AKT (1:5000) and anti-beta-actin (1:2000).
PVDF membranes were washed three times with TBST and incubated for 1 hour at 20-35 ℃ with secondary antibody binding, followed by washing and chemiluminescent detection, and imaging using ImageQuant LAS 4000.
Fig. 7 schematically shows an evaluation of in vitro reduction of PD-L1 expression by a divalent metal cation compound in example 1 of the present disclosure, and as shown in fig. 7, it can be seen from the experimental results that the divalent metal cation compound can reverse the increase in PD-L1 expression, even reduce the expression level of PD-L1 to a level lower than that of the control group. At the same time, p-AKT expression was down-regulated, while total AKT protein levels were unchanged. Thus, PS-AKT-PD-L1 axis can be activated and enhanced by chemotherapy-induced cancer cell surface phosphatidylserine eversion, while disruption of this signaling axis by divalent metal cation compounds can result in reduced PD-L1 expression.
The divalent metal cation compound obtained in example 1 was tested for in vivo antitumor immunotherapy, and the specific test procedure and evaluation results are as follows.
Wherein, all animal experiments are carried out according to guidelines of laboratory animal guidelines. In a therapeutic CT26 tumor model, CT26 cells were inoculated into BALB/c mice.
When the tumor volume of CT26 was close to 80mm 3, mice were divided into 4 groups, PBS group (1), CPT (3 mg/kg) group (2), CPT (3 mg/kg) +αPD-L1 (5 mg/kg) group (3) and CPT (3 mg/kg) +divalent metal cation compound (10 mg/kg) group (4), respectively. CPT was intravenously injected into the tail of the mice, and after 2 hours of intravenous injection, alpha PD-L1 or a divalent metal cation compound was injected into the tail of the mice, once every 1 day, for a total of three treatments. Tumor volume and weight were measured every 2 days to evaluate the antitumor therapeutic effect of the metal cation compound.
The calculation formula for tumor volume (V) is v=0.5×l×w 2, where L and W represent length and width, respectively. Tumors and lymph nodes were collected on day 5 after 3 cycles of treatment. T lymphocytes were isolated with a lymphocyte isolate and stained with FITC anti-mouse CD3, APC anti-mouse CD4and PerCP/cyanine5.5 anti-mouse CD 8. Alpha. Antibody, and then examined for intratumoral infiltration of T lymphocytes by flow cytometry.
Tumor draining lymph nodes were stained with PerCP/cyanine5.5 anti-CD11c, FITC anti-CD80AND APC ANTI-CD86 antibody, and DC maturity in tumor draining lymph nodes was then detected by flow cytometry.
Fig. 8 schematically shows the in vivo antitumor immunotherapeutic effect of the divalent metal cation compound in this example 1. As shown in fig. 8, wherein fig. 8a-c schematically show the change in tumor volume, mouse weight and survival rate after intravenous injection of 4 groups of mice, wherein the divalent metal cation compound treatment resulted in rapid and stable tumor regression, and the survival rate of mice was as high as 70%, showing good in vivo anti-tumor effect. Figures 8d-i schematically show the in vivo anti-tumor immunotherapeutic effect of divalent metal cation compounds, which can be seen to be effective in inducing DC maturation and further enhancing MHC II expression, facilitating antigen presentation. After intravenous injection treatment, significantly more CD4 +、CD8+ T cells were present in the tumor than in the other groups, indicating that the metal cation compound promoted enrichment of T cells in the tumor cells.
Example 2
A process for the preparation of a divalent metal cation compound comprising the steps of:
(1) To a 100mL flask were added terpyridine (100 mg,0.36 mmol), dicyclohexylcarbodiimide (DCC) (112 mg,0.54 mmol), and 4-Dimethylaminopyridine (DMAP) (13 mg,0.11 mmol), and dissolved in 5mL of DMF. Hexaethylene glycol (509 mg,1.81 mmol) was dissolved in DMF.
(2) The hexaethylene glycol solution was added to 5mL of DMF reaction solution over 30min with a constant pressure dropping funnel and reacted overnight at 20 ℃. After stirring overnight, the mixture was cooled and the solvent was removed under vacuum. The crude product was purified by column chromatography (dichloromethane: methanol=15:1) to give the metal cation compound precursor as a white solid. Fig. 9 schematically shows a nuclear magnetic resonance hydrogen spectrum of a metal cation compound precursor in this example 2, and the molecular formula of the compound precursor can be obtained as shown in fig. 9.
(3) 0.21Mmol of the metal cation compound precursor was dissolved in methanol, and a methanol solution in which zinc nitrate (0.21 mmol) was dissolved was added. The mixed solution is sonicated at 20-35 ℃ for 60min, followed by removal of the solvent under vacuum, and finally dried to give the divalent metal cation compound of formula (E).
The divalent metal cation compound obtained in example 2 was tested for specific binding property to phosphatidylserine, and specific test procedures and evaluation results are as follows.
The binding of divalent metal cation compound to phosphatidylserine was measured by flow cytometry experiments in which 4T1 cells in the logarithmic growth phase were digested with pancreatin and made into cell suspensions, 1x 10 5 cells were added per well in 12 well plates, after incubation for 12h, 4T1 cells were pretreated with 7.0 μm Camptothecine (CPT) for 24h to induce more phosphatidylserine exposure on the tumor cell surface, and PS + cells were obtained. PS + cells were harvested and incubated with varying concentrations of divalent metal cation compound to bind to phosphatidylserine on the surface of CPT-induced PS + cells. The FITC-labeled annexin (annexin v-FITC) in the apoptosis detection kit was then used to specifically bind to phosphatidylserine, and after staining was completed, the fluorescence intensity of FITC-positive cells in each group of cells was analyzed by Flow Cytometry (FCM) on FITC channels, and the bound amount of phosphatidylserine was detected.
FIG. 10 schematically shows an evaluation of specific binding between a divalent metal cation compound and phosphatidylserine in example 2. It should be noted that the number of the substrates, PS represents phosphatidylserine in the figure. As shown in fig. 10, more leaf phosphatidylserine eversion was promoted by chemotherapy drug camptothecin induction, and cancer cells (PS + cells) with stronger PS positive signals were obtained. PS positive cells incubated subsequently with different concentrations of divalent metal cation compound reduce PS exposure to varying degrees and bind to divalent metal cation compound, and bound PS is no longer able to continue to bind FITC fluorescent-labeled annexin (annexin v), resulting in reduced annexin v fluorescence, indicating that the divalent metal cation compound of formula (E) is not only able to specifically bind to PS, but is also able to mask PS interactions with other proteins or receptors.
While the foregoing is directed to embodiments of the present disclosure, other and further details of the invention may be had by the present application, it is to be understood that the foregoing description is merely exemplary of the present disclosure and that no limitations are intended to the scope of the disclosure, except insofar as modifications, equivalents, improvements or modifications may be made without departing from the spirit and principles of the present disclosure.
Claims (9)
1. Use of a divalent metal cation compound for the manufacture of a medicament for immunomodulation and anti-tumor by binding to phosphatidylserine on the surface of tumor cells, wherein the divalent metal cation compound has the structural formula shown in formula (I):
Formula (I)
Wherein M is selected from any one of Cu 2+、Zn2+、Ni2+;
r 1 is selected from single bonds;
x is selected from O;
R 2 is selected from Or (b)。
2. The use according to claim 1, wherein the divalent metal cation compound has a binding affinity constant for phosphatidylserine of 2.41-10.6x10 -6 M.
3. Use according to claim 1, wherein the divalent metal cation compound is suitable for inhibiting activation of the downstream AKT signaling pathway to inhibit PD-L1 expression.
4. A process for the preparation of a divalent metal cation compound for use in the application according to any one of claims 1 to 3, comprising the steps of:
reacting the compound (A) with the compound (B) in an organic solvent to obtain a compound (C);
Formula (A) formula (B)
Reacting the compound (C) with a divalent metal salt in an organic solvent to obtain a compound shown in a formula (I);
formula (C).
5. The method according to claim 4, wherein the divalent metal salt comprises at least one of a nickel salt, a copper salt, and a zinc salt.
6. The preparation 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 reacting the compound (a) and the compound (B) in an organic solvent comprises:
reacting the compound (A) and the compound (B) in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine under an inert gas atmosphere.
8. The production process according to claim 7, wherein the reaction temperature is 20 to 50 ℃ and the reaction time is 2 to 24 hours;
The reaction temperature of the compound (C) and the divalent metal salt is 20-40 ℃ and the reaction time is 1-24 hours.
9. A divalent metal cation compound suitable for use in any one of claims 1 to 3, said divalent metal cation compound having the structural formula (I):
Formula (I)
Wherein M is selected from any one of Cu 2+、Zn2+、Ni2+;
r 1 is selected from single bonds;
x is selected from O;
R 2 is selected from Or (b)。
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