阅读说明：本技术 一种具有抗淋巴细胞白血病功效的和厚朴酚-苯丁酸氮芥共前体药物及其制备方法和应用 (Honokiol-chlorambucil co-prodrug with lymphocyte leukemia resisting effect and preparation method and application thereof ) 是由 夏黎 汪小根 张雷红 沈小钟 张树潘 李绍林 于 2020-07-20 设计创作，主要内容包括：本发明属于抗癌药物技术领域,公开了一种具有抗淋巴细胞白血病功效的和厚朴酚-苯丁酸氮芥共前体药物及其制备方法和应用。所述共前体药物具有如式(Ⅰ)所示结构。制备方法包括以下步骤：将苯丁酸氮芥溶解于N,N-二甲基甲酰胺中,再加入N-乙基-N’-(3-二甲基氨基丙基)碳二亚胺盐酸盐,然后在室温下将溶液搅拌10min；然后加入和厚朴酚,并在室温下将反应混合物搅拌过夜；将反应液加入乙酸乙酯,然后用水洗涤,用硫酸钠干燥,过滤并浓缩,经色谱法纯化得到和厚朴酚-苯丁酸氮芥共前体药物。<Image he="522" wi="700" file="DDA0002592845410000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention belongs to the technical field of anti-cancer drugs, and discloses a honokiol-chlorambucil co-prodrug with an anti-lymphocyte leukemia effect, and a preparation method and application thereof. The co-prodrug has a structure shown as a formula (I). The preparation method comprises the following steps: dissolving chlorambucil in N, N-dimethylformamide, adding N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring the solution at room temperature for 10 min; then adding honokiol and stirring the reaction mixture at room temperature overnight; the reaction solution was added to ethyl acetate, washed with water, dried over sodium sulfate, and filteredFiltering, concentrating, and purifying by chromatography to obtain honokiol-chlorambucil co-prodrug.)

1. A honokiol-chlorambucil co-prodrug having anti-lymphocytic leukemia efficacy, comprising: the co-prodrug has a structure shown in the following formula (I):

2. the process for the preparation of honokiol-chlorambucil co-prodrug having anti-lymphocytic leukemia effect as claimed in claim 1, comprising the following steps: dissolving chlorambucil in N, N-dimethylformamide, adding N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring the solution at room temperature for 10 min; then adding honokiol and stirring the reaction mixture at room temperature overnight; the reaction solution was added to ethyl acetate, then washed with water, dried over sodium sulfate, filtered and concentrated, and purified by chromatography to give honokiol-chlorambucil co-prodrug.

3. The use of the honokiol-chlorambucil co-prodrug having anti-lymphocytic leukemia effect according to claim 1 in the preparation of an anti-lymphocytic leukemia medicament.

Technical Field

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a honokiol-chlorambucil co-prodrug with an anti-lymphocyte leukemia effect, and a preparation method and application thereof.

Background

Chlorambucil (CBL) is a DNA alkylating agent, belongs to the nitrogen mustard family, and is a chemotherapeutic drug for treating various solid tumors such as Chronic Lymphocytic Leukemia (CLL) and lymphoma. The N, N-bis (2-chloroethyl) -amine moiety can covalently react with proteins, nucleic acids and phospholipids to induce a cytostatic function in cell survival, whereas the alkylation reaction of CBL with DNA is the main form of cytotoxicity. Forms of CBL-modified DNA cross-linking include single-functional base pair mismatches and bifunctional double-stranded DNA breaks, leading to sustained DNA damage. Because CBL has high reactivity with a plurality of biological macromolecules (nucleic acid, protein and phospholipid), the clinical treatment effect is poor, the half life is short, and the CBL dosage required by the treatment response is high. However, increased dosages increase the risk of serious side effects. Moreover, the combined result of these instabilities and non-specific reactivity will reduce the biological rate of action of the CBL. Currently, while some new drugs have been successfully developed for clinical use, CBL remains in fact the first line treatment for elderly CLL and some immunosuppressed cancer patients. Therefore, the development of new CBL derivatives with high antitumor activity and stable toxicity has important significance on normal healthy tissues.

Honokiol (HN, C)₁₈H₁₈O₂) Is a natural product of dietary bisphenol separated from magnolia officinalis. In the last decade, a great deal of research shows that HN has a wide inhibition effect on malignant tumors (such as myeloma and leukemia) in vitro and in vivo through activities of resisting cancer, promoting apoptosis, resisting inflammation, resisting oxidation, resisting angiogenesis and the like, and has no obvious sub-toxicity. In addition, HN effectively inhibits the antiproliferative effects of multiple pathways and targets on cancer cells, such as NF-kB, EGFR, STAT3, cyclooxygenase and other cellsApoptosis factor, etc., and HN can also treat well-known drug-resistant tumors. HN is considered an anti-tumor drug comparable to the common chemotherapeutic drug Doxorubicin (DOX). Importantly, HN could target cancer cell mitochondria through STAT3, preventing tumor progression and metastasis, suggesting that HN may be a new effective chemopreventive or therapeutic entity for tumor treatment. However, clinical studies on HN are not yet common.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide a honokiol-chlorambucil co-prodrug (HN-CBL) with anti-lymphocytic leukemia effect.

The invention also aims to provide a preparation method of the honokiol-chlorambucil co-prodrug with the effect of resisting the lymphocytic leukemia.

Still another object of the present invention is to provide the use of the honokiol-chlorambucil co-prodrug having anti-lymphocytic leukemia effect.

The purpose of the invention is realized by the following technical scheme:

a honokiol-chlorambucil co-prodrug having anti-lymphocytic leukemia activity, the co-prodrug having the structure shown in formula (i):

the preparation method of the honokiol-chlorambucil co-prodrug with the effect of resisting the lymphocytic leukemia comprises the following operation steps: dissolving chlorambucil in N, N-dimethylformamide, adding N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring the solution at room temperature for 10 min; then adding honokiol and stirring the reaction mixture at room temperature overnight; the reaction solution was added to ethyl acetate, then washed with water, dried over sodium sulfate, filtered and concentrated, and purified by chromatography to give honokiol-chlorambucil co-prodrug.

The application of the honokiol-chlorambucil co-prodrug with the effect of resisting the lymphocytic leukemia in preparing the medicines for resisting the lymphocytic leukemia is provided.

The principle of the invention is as follows:

based on the molecular mechanistic background of Chlorambucil (CBL) and Honokiol (HN), the present inventors believe that the development of new anti-tumor agents from approved therapeutic drugs or safe dietary natural products, but not other unknown compounds, will facilitate their transformation and use in cancer therapy. The invention designs and synthesizes honokiol-chlorambucil (HN-CBL) ester co-prodrug, the HN-CBL is combined through carbonate ester bond, the release reaction mechanism of the HN-CBL is dicarbonate coupled with HN and CBL, the cell lysis can be simply hydrolyzed under the catalysis of higher intracellular esterase (such as cancer), and the honokiol-chlorambucil co-prodrug is particularly sensitive to tumor acid microenvironment (pH 5.5vs pH 7.4). When an in-vitro MTT cytotoxicity method is used for evaluating the inhibition effect of HN-CBL on a series of cancer cells and normal cell lines, the HN-CBL has better treatment effect than the maternal drugs HN and CBL by directly enhancing the mitochondrial activity. HN-CBL selectively enhances killing of Lymphocytic Leukemia (LL) cells, and no erythrocytic hemolytic reaction is observed at therapeutic concentrations. In addition, HN-CBL significantly promoted LLs apoptosis, but was not damaged by normal PBMCs. Computational docking and western-blotting studies have shown that HN-CBL also binds to STAT3 protein at certain hydrophobic residues and down-regulates the phosphorylation level of STAT3 protein. In conclusion, HN-CBL can obviously delay the growth of leukemia cells in vivo and has no obvious physiological toxicity. These results indicate that HN-CBL may provide a novel prodrug for selective treatment of LLs with fewer side effects than the free drug.

Compared with the prior art, the invention has the following advantages and effects:

HN-CBL can obviously inhibit the proliferation capacity of human leukemia cell strains CCRF-CEM, Jurkat, U937, MV4-11 and K562; in addition, HN-CBL can selectively inhibit the survival of lymphocyte leukemia cells, enhance the mitochondrial activity of the leukemia cells and induce LLs apoptosis; molecular docking and western-blot studies show that HN-CBL can also be combined with STAT3 protein on certain hydrophobic residues to reduce the phosphorylation level of STAT3 protein; HN-CBL can obviously delay the growth of leukemia cells in vivo and has no obvious physiological toxicity. Therefore, HN-CBL may provide a new and effective target treatment method for the target treatment of the lymphocytic leukemia.

Drawings

FIG. 1 is a graph of in vitro targeted release pharmacokinetics of HN-CBL in tumor cells, where A is the hydrolysis rate of HN-CBL in normal isotonic buffer PBS at different pH values, and B is the hydrolysis rate of HN-CBL in different biological media.

FIG. 2 shows the antiproliferative properties of HN-CBL and the therapeutic results of primary lymphocytic leukemia cells.

FIG. 3 is a graph of HN-CBL vs. mt 'superoxide levels and its membrane potential, where A is a graphical representation of increased superoxide concentration and B is a graphical representation of decreased mt' membrane potential.

FIG. 4 is a graph showing the comparison of HN-CBL, HN and CBL induced apoptosis in lymphocytic leukemia cells.

FIG. 5 is a comparison of HN, CBL and HN-CBL inhibition of tumor growth in vivo.

FIG. 6 is a pathological comparison of HN, CBL and HN-CBL inhibiting tumor growth in vivo.

FIG. 7 is a chart of hemolysis experiment of HN-CBL.

FIG. 8 is a graph showing the results of flow cytometry analysis of HN-CBL effects on peripheral blood lymphocytes from healthy donors and lymphocytes from lymphoblastic leukemia patients.

Fig. 9 is a pathological section of the main organs of differently treated mice.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Reagents and instruments used in the following examples: chlorambucil (CBL, HPLC purity)

>95%), honokiol (HN, HPLC purity)>95%), N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO)Ethyl acetate and sodium sulfate were purchased from shanghai energy and chemical company, china. Chromatographic purity Acetonitrile (ACN) was from siemer feishel. MTT (98%) was from MedChemExpress (shanghai, china). The apoptosis detection kit for Annexin V-FITC and other cell assay reagents are available from Life Gibco Technologies, Inc. of Grand Island, Saimer Feishale, USA. Antibodies to p-STAT3(#9134S), STAT3(9139S), and actin were purchased from Cell Signaling Technology (Denver, Mass.). Deuterated chloroform (CDCl) was recorded on a Bruker AM-400 NMR spectrometer₃) 1H-NMR spectrum in (1). Chemical shifts are expressed in (ppm) as internal reference compared to Tetramethylsilane (TMS). High Resolution Mass Spectra (HRMS) were recorded on an Agilent model 1260 UPLC-Waters Q-TOF microspectrometer. The change of the release amount of HN-CBL along with the mass-to-charge ratio of molecular ions is measured by adopting an Agilent 1260 ultra performance liquid chromatography C18 column and an ultraviolet-visible spectrophotometer.

Cell culture and buffer conditions used in the following examples: CCRF-CEM, Jurkat, U937, MV4-11, K562 and LO2 cells were purchased from ATCC (Bessesda, Md., USA) or CTCC (Shanghai, China, CAS), cultured in DMEM or RPMI1640 medium (Gibco), and supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 100 units of antibiotic (Gibco) at 5% CO₂Purification of LL leukemia patients (sub-types according to the FAB classification system) and healthy human Peripheral Blood Mononuclear Cells (PBMCs) with Ficoll buffer in Ficoll isolation of peripheral blood from healthy donors erythrocytes were also obtained, mononuclear cells were suspended in RPMI1640 medium containing 20% FBS at a concentration of about 5-10 × 10⁵and/mL. With no Ca content²⁺、Mg²⁺The cells were washed with Dulbecco's phosphate buffer (DPBS, Invitrogen).

The clinical biological specimen collection and hemolysis assay methods used in the following examples: peripheral blood lymphocytes from healthy donors were purified and named PBMCs and lymphocytes from lymphoblastic leukemia patients were named LLs according to protocols approved by the research ethics committee. Peripheral blood mononuclear cells were isolated by Ficoll centrifugation. Red blood cells were isolated from healthy donor blood and purified with chilled PBS. In etcCBL, HN and HN-CBL solutions were prepared in the lysis buffer, added to the erythrocyte buffer, and subjected to the blood lysis analysis on a 96-well plate. Briefly, 2. mu.L of red blood cells were added to each well, mixed, and then incubated at 37 ℃ in 5% carbon dioxide for 1 hour. For 100% lysis, 50% H was added to the wells₂O, for negative control (0%), only cells from these wells (including PBS buffer) were used-after centrifugation at 1000 × g for 10min, the supernatant was transferred to a new tube, PBS was added and mixed, read at 450nm to determine absorbance.

Cytotoxicity was determined using the MTT method in the following examples A549, HepG2, NIH3T3 and LO2 cells at 4-6 × 10³Density of cells/well in 96-well plates leukemia cell lines (CCRF-CEM, Jurkat, U937, MV4-11 and K562) at 10-20 × 10³The density of cells/well was seeded on 96-well plates. IC50 values and statistical analysis were performed using GraphPad Prism 5.01(GraphPad software).

The method for measuring superoxide in mitochondria used in the following examples, we shall refer to 4 × 10 according to the superoxide kit instructions (Invitrogen)⁴Individual leukemia cells were plated in 12-well plates to verify the effect of CBL, HN or HN-CBL on superoxide in the mitochondria of leukemia cells. After 1.5 hours incubation of the cell and drug mixture, we first removed the media, then completed the trypsinization and centrifugation process, and the resulting cells were stained with MitoSOX and used with a BD-facsverse (flow cytometer) (BD Biosciences, annaburg, michigan).

The apoptosis assay in the following examples is specifically: apoptosis of PBMCs and LLs cells was detected 24 hours after treatment with or without HN, CBL and HN-CBL, respectively, with the aid of a BD-FACSVerseM flow cytometer (BD-Biosciences, CA). The differences between untreated and HN, CBL or HN-CBL treatment were compared with the reference to the specification of the Benetimine V-FITC/PI apoptosis kit for Beyotime (China) using PBS as the target cell.

The following examples illustrate the molecular docking of HN-CBL co-prodrugs with HN: molecular ligand docking studies were performed with Sybyyl-x2.1.1 software that minimizes energy using default parameters. The X-ray crystal structure of STAT3(PDB code: 3CWG) was obtained from the protein database. Docking is performed with the docking pod sized large enough to include the binding location.

The immunoblotting method used in the following examples specifically followed the following steps: lymphocytic leukemia cells were treated with 10. mu. MHN or HN-CBL for 12 hours, and the cells collected were lysed in 200. mu.L of WB and IP lysis buffer (1% Triton X-100) containing 1mM PMSF (Beyotime, China). The protein extracts (50. mu.g) were loaded onto 8-15% polyacrylamide gels containing SDS, electrophoresed and transferred to 0.22 μm nitrocellulose membranes (PALL, USA). Membranes were blocked with 5% skim milk powder in Tris buffered saline containing 0.1% Tween 20(TBST) and incubated overnight at 4 ℃ with primary antibody. Washed three times with TBST and detected with HRP conjugated secondary antibody for 2h at room temperature. The immune complexes were visualized using the Photope-HRP Western-Blot detection system (Pierce, USA). Actin is used to ensure an equivalent load of whole cell proteins. All data were confirmed by three separate experiments.

The in vivo xenograft model used in the following examples was specifically prepared according to the following procedure female BALB/c nudes (4-6 weeks old) were taken from the center of laboratory animals (Changsha) of Hunan province, all animal studies were conducted according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of traditional Chinese medicine of Hunan province, and CCRF-CEM cells (1.2 × 10) were cultured 5 days after the mice were acclimatized to the new environment⁷0.2 ml/mouse) was injected subcutaneously into the flank (day 0). When the tumor volume reaches 100mm³On the left and right, mice were randomized into 4 groups, injected intravenously with HN (3.5mg/kg), CBL (8mg/kg) and HN-CBL co-prodrug (11mg/kg, equivalent to HN dose 3.5mg/kg or 8mg/kg CBL) every two days, tumor volume (V) and mouse body weight were recorded every three days using the formula V ═ a × b (a × b)²) The/2 calculation, where "a" and "b" represent the length and width of the tumor diameter, respectively. After the experiment was completed, mice were sacrificed on day 24, and tumors and major organs were taken, fixed in formaldehyde, and paraffin-embedded.

The following examples of tumor tissue TUNEL apoptosis detection, Ki67 proliferation assay and major organ H & E staining specifically employ the following methods: tumor tissue apoptosis was detected using TUNEL detection kit (Roche). Briefly, tumor tissue from paraffin-embedded specimens was deparaffinized in xylene and rehydrated with reduced concentrations of ethanol. Cell proliferation in tumor tissues was detected by labeled streptavidin-biotin immunohistochemistry. The morphology of the major organs was observed by hematoxylin-eosin staining.

The statistical analysis described in the following examples is specifically as follows: for data analysis, values from independent experiments are presented as mean ± SEM. Statistical differences Using non-spectral Student's two-tailed t test, p <0.05 was considered statistically significant.