Nitrogen mustard active probes and their applications

Abstract

The present invention provides a nitrogen mustard active probe and its application. The probe comprises a compound having the structure shown in the following formula. The present invention further provides a method for synthesizing the nitrogen mustard active probe and its application in preparing a nitrogen mustard marker or anti-tumor drug. Furthermore, a nitrogen mustard marker or anti-tumor drug containing the nitrogen mustard active probe as an active component is provided. The probe of the present invention can be used as a bioorthogonal labeling probe, particularly for labeling nitrogen mustard in tissues and cells, and for studying the toxicological mechanisms of nitrogen mustard in anti-tumor and immunosuppressive effects. The probe can be used to explore the direct protein targets of nitrogen mustard, contributing to a deeper understanding of its mechanism of action. The active probe itself has certain anti-tumor and immunosuppressive effects and can be used as an immunosuppressive and / or anti-tumor drug.

Description

Technical Field

[0001] The present invention relates to the field of medical biological detection and pharmaceutical technology, and relates to a nitrogen mustard active probe and its application in nitrogen mustard intracellular labeling and anti-tumor. Background Art

[0002] Nitrogen mustard is an organic compound with the chemical formula C5H 11 C l2 Nitrogen mustard is the earliest anti-tumor drug used clinically with outstanding efficacy. It is a representative of the bischloroethylamine class of alkylating agents. It is the origin of nitrogen mustard anti-tumor chemotherapy drugs and can be used to treat malignant lymphoma, lung cancer, head and neck cancer, and Hodgkin's disease. It is also clinically used to treat psoriasis and lupus nephropathy. Nitrogen mustard is also a erosive agent that can cause local skin blistering and ulceration. Researchers have also developed a large number of nitrogen mustard drugs based on its structure, such as cyclophosphamide and semustine.

[0003] After entering the body, nitrogen mustard forms a highly reactive ethyleneimine ion through intramolecular cyclization. Under neutral or weakly alkaline conditions, it rapidly binds to nucleophilic groups on a variety of organic substances (such as carboxyl, amino, and sulfhydryl groups on proteins, amino and hydroxyl groups on nucleic acids, and phosphate groups), undergoing alkylation. The most important reaction of nitrogen mustard is covalent binding to the nitrogen at position 7 of guanine, generating cross-links within the DNA duplex or between different bases within the same DNA strand. Cells in the G1 and M phases are most sensitive to the cytotoxic effects of nitrogen mustard, and their transition from G1 to S phase is delayed.

[0004] Although studies have shown that nitrogen mustard can bind to various biomacromolecules in vivo, its specific protein targets and mechanisms are still unclear. Due to the lack of effective nitrogen mustard markers, it is difficult to determine the specific targets of nitrogen mustard in vivo. Summary of the Invention

[0005] In order to clarify the specific protein targets of nitrogen mustard in the body, the present invention has developed a nitrogen mustard activity probe that can be visualized in cells and tissues and can be used to identify target proteins. It also has strong anti-tumor activity and can be used to prepare corresponding drugs.

[0006] In a first aspect, the present invention provides a nitrogen mustard active probe, a compound having a structure shown in Formula I:

[0007]

[0008] Wherein, n is a positive integer between 0 and 5.

[0009] Preferably, n is 1 or 2.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned nitrogen mustard active probe, and the reaction structure is as follows:

[0011]

[0012] The synthesis process is as follows: di(2-chloroethyl)amine and bromoalkyne are added to an organic solvent, and reacted at 25-160° C. for 8-72 hours in the presence of an alkaline reagent to obtain the nitrogen mustard active probe.

[0013] Preferably, the organic solvent is selected from at least one of dichloromethane, acetonitrile, n-butanol, methanol, and dimethyl sulfoxide; and the alkaline reagent is selected from at least one of potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methoxide, and cesium carbonate.

[0014] The third aspect of the present invention provides the use of the nitrogen mustard activity probe, specifically its use in the preparation of nitrogen mustard markers or anti-tumor drugs.

[0015] Preferably, the nitrogen mustard marker is an intracellular nitrogen mustard marker, and the direct protein target of the nitrogen mustard is identified by fluorescent gel analysis, mass spectrometry-based target protein quantification and site identification. The anti-tumor drug is an anti-melanoma drug.

[0016] In a fourth aspect, the present invention provides a nitrogen mustard marker, the functional component of which is the nitrogen mustard active probe described above, which acts inside cells and realizes the identification of specific targets through tracking analysis.

[0017] In a fifth aspect, the present invention provides an anti-tumor drug comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the above-mentioned nitrogen mustard active probe.

[0018] The beneficial protection and effects of the present invention are as follows:

[0019] (1) Experimental results show that the nitrogen mustard active probe of the present invention can act directly inside cells, enabling tracking of nitrogen mustard inside cells through cell imaging. This probe can be used as a bioorthogonal labeling probe, particularly for labeling nitrogen mustard in tissues or cells, to study the pharmacological and toxicological mechanisms of nitrogen mustard in antitumor and immunosuppression, and to explore the direct protein targets of nitrogen mustard, thereby contributing to a deeper understanding of its mechanism of action.

[0020] (2) The inhibitory effect of the nitrogen mustard active probe on the viability of tumor cells, including HaCaT cells, is concentration-dependent, indicating that the active probe itself has a certain anti-tumor effect and can be used as an anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the H NMR spectrum of the nitrogen mustard active probe I-1;

[0022] Figure 2 This is the C NMR spectrum of the nitrogen mustard active probe I-1;

[0023] Figure 3 This is the H NMR spectrum of the nitrogen mustard active probe I-2;

[0024] Figure 4 This is the C NMR spectrum of the nitrogen mustard active probe I-2;

[0025] Figure 5 This is the fluorescence imaging result of nitrogen mustard active probe I-1 in cells;

[0026] Figure 6 This is the direct labeling result of nitrogen mustard active probe I-1 based on fluorescent gel analysis;

[0027] Figure 7 The inhibitory effect of different concentrations of nitrogen mustard active probe I-1 on HaCaT cell viability. DETAILED DESCRIPTION

[0028] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0029] All reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are calculated by volume.

[0030] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and each feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.

[0031] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0032] The numerical ranges herein include their endpoints and each specific numerical point and sub-range within the numerical range. For example, 1 to 3 includes endpoints 1 and 3, the specific integer numerical point 2 and non-integer numerical point therein (for example, but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (for example, but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0034] Example 1: Synthesis of active probe I-1

[0035]

[0036] 0.1 mmol of di(2-chloroethyl)amine and 0.1 mmol of bromopropyne were added to dichloromethane, and 0.05 mmol of potassium carbonate was added, and the mixture was reacted at 20°C for 20 h. The reaction solution was dried and separated by column chromatography (dichloromethane) to obtain an oily substance I-1 with a yield of 60.9%.

[0037] The hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum of the nitrogen mustard active probe I-1 are as follows: Figure 1 and Figure 2 shown.

[0038] Example 2: Synthesis of active probe I-2

[0039]

[0040] 0.1 mmol of di(2-chloroethyl)amine and 0.1 mmol of bromobutyne were added to acetonitrile, and 0.05 mmol of potassium carbonate was added, and the mixture was reacted at 80°C for 8 h. The reaction solution was spin-dried and separated by column chromatography (dichloromethane) to obtain an oily substance I-2 with a yield of 50.2%.

[0041] The hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum of the nitrogen mustard active probe I-2 are as follows: Figure 3 and Figure 4 shown.

[0042] Example 3: Application of Nitrogen Mustard Active Probe I-1 as a Nitrogen Mustard Labeled Probe in Cell Imaging

[0043] HaCaT cells were plated in culture dishes and grown to 80% of their cells before being divided into groups: blank, low-concentration probe group, medium-concentration probe group, and high-concentration probe group. 0.1% DMSO was added to the blank group, 10 μM I-1 was added to the low-concentration probe group, 50 μM I-1 was added to the medium-concentration probe group, and 100 μM I-1 was added to the high-concentration probe group. The cells were incubated for 2 hours.

[0044] 100 μM azidrhodamine, 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, and 1 mM copper sulfate were premixed and added to the cells for incubation. After the reaction was complete, PBS was added and washed three times. Cell nuclei were stained with DAPI. Fluorescence microscopy was used to photograph the results. Figure 5 , the whereabouts of nitrogen mustard can be effectively tracked.

[0045] Example 4: Direct labeling experiment for fluorescent gel analysis

[0046] Thaw skin tissue stored at -80°C on ice, add lysis buffer, grind, and ultrasonically disrupt. Centrifuge at 20,000g for 30 minutes at 4°C. Collect the supernatant, quantify the protein concentration, and dilute to a desired concentration for later use. Prepare HNP-1 according to Table 1 below:

[0047] Table 1 HNP-1 and its blocking solution configuration

[0048]

[0049] 0.5 μL of DMSO or HNP-1 was added to 50 μL of the proteomic solution and centrifuged to a final DMSO solution of 1%. The mixture was incubated at 25°C for 1 hour. TAMRA-azide, BTTAA / CuSO4, and sodium ascorbate were sequentially added to each sample for a click reaction. The mixture was shaken at 25°C for 1 hour. After the reaction, 13.75 μL of loading buffer was added to each sample, boiled, and cooled. 40 μL of each sample was subjected to 10% SDS-PAGE electrophoresis. Fluorescence signals were detected using ChemiDoc MP, and proteins were detected by staining.

[0050] According to the results of the fluorescent gel experiment, the labeling intensity increases with the increase of HNP-1 concentration. According to the comparison of the fluorescent band and the Coomassie brilliant blue band, there is a certain selectivity, which shows that HNP-1 can specifically label proteins, among which the labeling effect is obvious near 40-50 and 70kDa. The CBB signal of the yellow-marked protein is weakened. According to the results of three biological repetitions, it should be caused by the increase of the probe concentration, which leads to protein cross-linking and molecular weight migration. In order to quantitatively identify proteins with high reactivity to HNP-1, combined with the literature method (Quantitative reactivity profiling predictsfunctional cysteines inproteomes. Nature. 2010, 468 (7325): 790-5), 1 / 10 / 100μM probe concentrations were subsequently selected for mass spectrometry experiments. The labeling results of Rhodamine and CBB are shown in Figure 6 .

[0051] Example 5:

[0052] HaCaT cells were passaged and cultured using the trypsin digestion method containing 0.01% EDTA. When the cells occupied 80% of the effective area at the bottom of the flask, they were trypsinized and blown into a single-cell suspension. The cells were counted under a microscope and diluted to a suspension containing 100 cells per microliter. 100 μl of the cell suspension was inoculated into a 96-well plate per well, and the plates were cultured in a 37°C, 5% CO2 saturated humidity incubator. Experiments were performed when the cell density reached 80%.

[0053] The experiment set up a blank control group and different concentrations of nitrogen mustard active probe I-1 poisoning groups, with 3 replicates in each group. Different concentration gradients of mustard gas poisoning solution were prepared. Fresh culture medium was replaced 30 minutes after poisoning. After 24 hours of incubation in the cell culture incubator, 10μl CCK-8 solution was added to each well. After incubation in the incubator for 1.5 hours, the absorbance at 450nm was measured by microplate reader and the cell viability was calculated. The results are as follows Figure 7 The structure shows that the inhibitory effect of the nitrogen mustard active probe I-1 on cell viability is concentration-dependent, and the higher the concentration of the nitrogen mustard active probe I-1, the stronger the inhibitory effect on cell viability.

[0054] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of a nitrogen mustard activity probe in the preparation of an intracellular nitrogen mustard marker, characterized in that: The nitrogen mustard active probe has a compound having a structure shown in Formula I: Where n is 0 or 1.

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