CN115192564B

CN115192564B - Phenylalanine derivative, pharmaceutical composition and application thereof in tumor treatment

Info

Publication number: CN115192564B
Application number: CN202210564219.9A
Authority: CN
Inventors: 彭勇; 魏于全; 蒲文臣; 马虎林; 徐富滟
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-05-23
Filing date: 2022-05-23
Publication date: 2023-11-17
Anticipated expiration: 2042-05-23
Also published as: CN115192564A

Abstract

The invention discloses a phenylalanine derivative, a pharmaceutical composition and application thereof in tumor treatment, and relates to the technical field of antitumor pharmacy. A compound represented by the structural formula (1) below or a pharmaceutically acceptable salt thereof: formula (1):the phenylalanine derivative is easy to prepare and has good drug-forming property, and can activate the cancer-inhibiting p53 signal transduction at the cellular and animal level and inhibit the growth of tumor. Specifically, phenylalanine derivatives inhibit the ubiquitination-proteasome degradation process of p53 by regulating the interaction of wild-type p53 and HDM2, increase the stability of p53 protein, up-regulate p53 signal transduction, and inhibit tumor growth. The phenylalanine derivative has good application prospect in preparation and antitumor drugs.

Description

Phenylalanine derivative, pharmaceutical composition and application thereof in tumor treatment

Technical Field

The invention relates to the technical field of antitumor pharmacy, in particular to a phenylalanine derivative, a pharmaceutical composition and application thereof in tumor treatment.

Background

World health organization (World Health Organization, WHO) issues "2020 world health statistics report" (World Health Statistics 2020) that about 900 tens of thousands of people die from malignant tumors worldwide each year is one of the main causes of human death. Malignant tumors represented by lung cancer, colorectal cancer and liver cancer have clinical characteristics of high malignant degree and poor prognosis, and become a serious public health problem seriously threatening human health. Tumor evolution is a complex process involving multiple steps, pathways, and factors, involving dysfunctions in the multidimensional layers of DNA, RNA, proteins, etc. At present, the treatment of malignant tumors remains a worldwide problem.

p53 is a key protein for regulating apoptosis and cell cycle of tumor cells, and is an important cancer suppressor in the process of tumor evolution. Studies show that p53 is a target protein of an important oncogene HDM2, and that the HDM 2-mediated ubiquitination-proteasome degradation pathway can regulate the stability and abundance of p53 in cells. The HDM2-p 53-based molecular regulation network plays an important role in the malignant progression process of tumors, and small molecules capable of regulating p53 signal transduction need to be searched for, so that a new strategy is provided for clinical treatment of tumors.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a phenylalanine derivative, a pharmaceutical composition and application thereof in tumor treatment so as to solve the technical problems.

The invention is realized in the following way:

in the present specification, "tumor" refers to an uncontrolled, growing population of cells due to genetic variation, including benign tumors and malignant tumors. In this specification, the term "malignancy" is used interchangeably with the term "cancer". The term "carcinoma" broadly includes carcinomas of the blood system such as carcinomas, sarcomas, and leukemias as of epithelial origin. Examples of cancers of epithelial origin include: stomach cancer, large intestine cancer, gall bladder cancer, bile duct cancer, pancreatic cancer, duodenal cancer, kidney cancer, prostate cancer, ovarian cancer, uterine cancer, breast cancer, skin cancer, hepatocellular carcinoma, tongue cancer, esophageal cancer, throat cancer, and the like, but are not limited thereto. Examples of sarcomas include: fibrosarcoma, malignant fibrous histiocytoma, dermal fibrosarcoma, liposarcoma, myosarcoma, angiosarcoma, kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, osteosarcoma, etc., but is not limited thereto. Examples of hematological malignancies include: leukemia, malignant lymphoma, multiple myeloma, and the like, but is not limited thereto. In the present specification, "tumor cells" means cells that form a tumor, and typically refer to cells that proliferate abnormally (so-called cancerous cells) regardless of surrounding normal tissues.

The invention provides a phenylalanine derivative, which contains a compound shown in the structural formula (1) or pharmaceutically acceptable salt thereof:

formula (1):wherein R is ¹ 、R ² 、R ³ And R is ⁴ Each independently is hydrogen, aryl, acyl, carboxylic acid ester, sulfonic acid ester, sulfinic acid ester, nitrile, alkyl, alkenyl, alkynyl, halogen, alkoxy, amino, thioether, nitro, nitroso, substituted alkyl, substituted aryl, five membered heterocycle, six membered heterocycle, silicon, phosphonic acid or phosphonate.

The substituted alkyl is selected from the group consisting of halogen substituted alkyl, carboxy substituted alkyl, amino substituted alkyl, nitro substituted alkyl, and the like.

The substituted aryl is selected from the group consisting of halo-substituted aryl, carboxy-substituted aryl, amino-substituted aryl, nitro-substituted aryl, and the like.

The inventor finds that the phenylalanine derivative is easy to prepare and good in drug-forming property, and can activate cancer-inhibiting p53 signal transduction at the cellular and animal level and inhibit tumor growth. Specifically, phenylalanine derivatives inhibit the ubiquitination-proteasome degradation process of wild-type p53 by regulating the interaction of wild-type p53 and HDM2, increase p53 protein stability, up-regulate p53 signal transduction, and inhibit tumor growth.

In other embodiments, the above-described compounds useful as medicaments are pharmaceutically acceptable salts, "pharmaceutically acceptable salts" include pharmaceutically acceptable inorganic or organic acid salts.

Alternatively, the inorganic acid salt may be a sulfate, sulfite, hydrochloride, hydrobromide, nitrate, phosphate or dihydrogen phosphate salt.

Alternatively, the inorganic acid salt may be a hydrohalic acid salt, a halogen series oxygen-containing inorganic acid salt, a carbon, nitrogen, boron series oxygen-containing inorganic acid salt, a phosphorus series inorganic acid salt, a sulfur series inorganic acid salt, a hydrocyanic acid salt, a chromate, a dichromate, a manganate, a permanganate, a molybdate or a tungstate.

The hydrohalate may be hydrofluoric acid salt, hydrochloride salt, hydrobromide salt, hydroiodide salt, or the like.

The halogen series oxygen-containing inorganic acid salt can be hypochlorite, chlorate, perchlorate, hypobromite, bromate, hypoiodite, iodate or periodate, etc.

The oxygen-containing inorganic acid salts of carbon, nitrogen and boron can be carbonate, nitrite, nitrate, nitrite, borate, (original) borate, perborate, metasilicate or (original) silicate.

The inorganic acid salt of phosphorus series may be metaphosphite, (n) phosphate, phosphite, pyrophosphate, pyrophosphite, hypophosphite, per (mono) phosphate, per (di) phosphate or even (mono) phosphate, etc.

The sulfur series inorganic acid salt can be hydrogen sulfate, sulfite, sulfate, persulfate (mono) sulfate, thiosulfate, dithionate, pyrosulfate, peroxodisulfate, etc.

Alternatively, the organic acid salt may be acetate, maleate, fumarate, succinate, citrate, p-toluenesulfonate, tartrate, formate, propionate, heptanoate, oxalate, benzoate, malonate, succinate, maleate, hydroxybutyrate, citrate, methanesulfonate, benzenesulfonate, lactate or mandelate.

Alternatively, the organic acid salt may be an organic acid salt compound having a functional group such as a carboxyl group, a sulfonic acid group, a sulfinic acid group, or a thiocarboxylic acid group.

In other alternatives, the above-described inorganic acid salts and organic acid salts are not limited in scope by the present invention.

In a preferred embodiment of the present invention, the alkyl group is at least one selected from cycloalkyl and non-cycloalkyl groups;

in an alternative embodiment, the cycloalkyl is selected from C _3-8 Cycloalkyl groups. For example selected from: c (C) ₃ ，C ₄ ，C ₅ ，C ₆ ，C ₈ Cycloalkyl groups.

In an alternative embodiment, the non-cycloalkyl group is selected from C ₁ -C ₈ An alkyl group.

The alkenyl group is selected from the group consisting of-C ₁ -C ₈ Alkenyl, -C ₃ -C ₈ -a cycloalkenyl group. Alkynyl is selected from the group consisting of-C ₂ -C ₈ Alkynyl groups.

In a preferred embodiment of the present invention, the amine group is at least one selected from the group consisting of primary amine, secondary amine, tertiary amine, amide group, sulfonate group and sulfinate group.

In an alternative embodiment, the phenylalanine derivative has the structural formula of any one of:

the invention also provides a pharmaceutical composition which comprises the phenylalanine derivative.

In an alternative embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable additives or adjuvants.

Alternatively, the pharmaceutical composition dosage form is selected from the group consisting of tablets, pills, powders, suspensions, aqueous or oily solutions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, nasal sprays, aerosols, sprays, injections, sterile aqueous or oily solutions or suspensions for parenteral (including intravenous, intramuscular or infusion) or sterile emulsions.

In other embodiments, the pharmaceutical composition dosage form may be other pharmaceutically acceptable dosage forms, and is not limited to the above-described protective scope.

In an alternative embodiment, a liquid formulation may be prepared using sterile water or water-propylene glycol solution as a solvent, and the active ingredient may also be formulated in an aqueous polyethylene glycol solution. Aqueous solutions for oral administration may be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickeners as desired. Aqueous suspensions for oral use can be prepared by dispersing the finely divided active component in water together with viscous materials such as natural synthetic gums, resins, methylcellulose, carboxymethylcellulose and other suspending agents known in the pharmaceutical arts.

The pharmaceutical composition may be in unit dosage form. In these forms, the composition is divided into unit doses containing appropriate amounts of the active component. The unit dosage form may be a packaged formulation, the package containing discrete amounts of the formulation, such as a boxed tablet, capsule, and powder in a vial. The unit dosage form may also be a capsule, cachet or tablet or it may be the appropriate number of any of these packaged forms.

The invention also provides application of the phenylalanine derivative or the pharmaceutical composition in preparing antitumor drugs.

In a preferred embodiment of the present invention, the anti-tumor drug is a p53 signaling drug.

In a preferred embodiment of the invention, the tumor is selected from malignant tumors;

in an alternative embodiment, the tumor is selected from liver cancer, breast cancer, non-small cell lung cancer, cervical cancer, colon cancer, melanoma, lung cancer, ovarian cancer, gastric cancer, renal cancer, or bladder cancer.

In an alternative embodiment, the tumor is selected from liver cancer, breast cancer, non-small cell lung cancer, cervical cancer or colon cancer.

In an alternative embodiment of the present invention, the tumor is selected from lung cancer, breast cancer, colon cancer, midline cancer, interstitial tumor, liver tumor, renal cancer, neuroma, adrenal cancer, acinar cell carcinoma, acoustic neuroma, acrofreckle nevus melanoma, acrosweat adenoma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia (acute myeloid leukemia), acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue tumor, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, acinar soft tissue sarcoma, amethyoid fibroma, anaplastic large cell lymphoma, thyroid undifferentiated carcinoma vascular immunoblastic T-cell lymphoma, vascular myolipoma, vascular sarcoma, astrocytoma, atypical teratoma rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, blastoma, bone carcinoma, brennar tumor, bronzoma, burkitt lymphoma, brain carcinoma, cancer cells, carcinoma in situ, carcinoma sarcoma, chondroma, cementoma, myelema, chondrioma, chordoma, choriocarcinoma, chorioallantoic papilloma, clear cell sarcoma of the kidney, craniopharyngeal tube tumor, cutaneous T-cell lymphoma, cervical carcinoma, colorectal carcinoma, degos's disease, connective tissue-promoting small round cell tumor, diffuse large B-cell lymphoma, embryogenic dysplastic neuroepithelial tumor, anaplastic, embryonal carcinoma, endocrine gland tumor, endoblastoma, enteropathy-associated T-cell lymphoma, esophageal carcinoma, fetuses, fibromas, fibrosarcomas, follicular lymphomas, follicular thyroid carcinoma, gangliomas, gastrointestinal cancers, germ cell tumors, gestational choriocarcinomas, giant cell fibroblasts, bone giant cell tumors, gliomas, glioblastomas, gliomas, brain gliomas, glucagon tumors, gonadal blastomas, granulomatous tumors, male cell tumors, gall bladder cancer, gastric cancer, hairy cell leukemia, angioblastomas, head and neck cancers, vascular epidermoid tumors, malignant blood diseases, hepatoblastomas, hepatosplenic T-cell lymphomas, hodgkin's lymphomas, non-hodgkin's lymphomas, invasive lobular carcinomas, intestinal cancers, renal cancers, laryngeal carcinomas, freckle-like nevi, fatal midline cancers leukemia, testicular stromal cell tumor, liposarcoma, lung cancer, lymphoangioma, lymphosarcoma, lymphoepithelial tumor, lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant salamander, mantle cell lymphoma, marginal zone B cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, breast medullary carcinoma, thyroid medullary carcinoma, medulloblastoma, melanoma, meningioma, mercker cell carcinoma, mesothelioma, metastatic urothelial carcinoma, miao Leguan mixed tumor, myxoma, multiple myeloma, muscle tissue tumor, mycosis fungoides, myxosarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neuroblastoma, neurofibromatosis, neuroma, nodular melanoma, ocular cancer, oligodendrocytoma, oligodendroglioma, eosinophilic tumor, optic-sheath meningioma, optic neuroma, oral cancer, osteosarcoma, ovarian cancer, pankostema, papillary thyroid carcinoma, paraganglioma, pineal blastoma, pineal somatic tumor, pituitary cytoma, pituitary adenoma, pituitary tumor, plasmacytoma, multiple blastomas, precursor T-lymphocyte lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, primary peritoneal carcinoma, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma peritoneum, renal cell carcinoma, nephromyelitis carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, transfer carcinoma of Richst, rectal cancer sarcomas, schwannomosis tumor, seminomas, testicular supportive cell tumor, sex cord-gonadal stromal tumor, ring cell carcinoma, skin carcinoma, small blue cell tumor, small cell carcinoma, soft tissue sarcoma, somatostatin tumor, smoke wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, cerclash disease, small intestine tumor, squamous cell carcinoma, gastric cancer, T cell lymphoma, testicular cancer, follicular cell tumor, thyroid carcinoma, transitional cell carcinoma, throat carcinoma, umbilical duct carcinoma, genitourinary carcinoma, urothelial carcinoma, eye pigment layer melanoma, uterine carcinoma, warty carcinoma, visual pathway glioma, vulval carcinoma, vaginal carcinoma, waldenstrom macroglobulinemia, wo Xinliu, and wilms tumor.

The invention also provides application of the phenylalanine derivative or the pharmaceutical composition in preparing a tumor treatment drug, which is used for:

inhibit the ubiquitination-proteasome degradation process of p 53; increase p53 protein stability or up-regulate p53 signaling.

The present invention also provides a pharmaceutical composition comprising: phenylalanine derivatives or the pharmaceutical compositions and combination drugs.

In the pharmaceutical composition of the present invention, the active ingredient may be only the compound of the present invention, or may be combined with other antitumor compounds as the active ingredient.

In an alternative embodiment, the combination includes, but is not limited to: chemotherapy drugs, radiotherapy, photosensitizers, photothermal agents, immunotherapy and the like.

The combination drug is at least one of the following drugs: antiproliferative or antineoplastic agents for medical oncology, cytostatic agents, antiinvasive agents, inhibitors of growth factor function, antiangiogenic agents or vascular damaging agents.

Antitumor agents such as: folic acid antagonists, antimetabolites, alkylating agents, platinum salts, anthracyclines and intercalating agents, antimtopoisomerase, cytoskeletal agents, bleomycin, asparaginase, disulfiram and mixtures thereof.

The antineoplastic agent is selected from the group consisting of methotrexate, 5-fluorouracil, fluorodeoxyuracil, cytarabine, 6-mercaptopurine, 6-thioguanine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, mitomycin C, aziridinyl benzoquinone (AZQ), busulfan, carmustine (BCNU), lomustine (CCNU), fotemustine, carboplatin, daunorubicin, doxorubicin or doxorubicin, epirubicin, dactinomycin or actinomycin D, mitoxantrone, amsacrine, podophylloguanide, etoposide, irinotecan, topotecan, vincristine, vinblastine, vindesine, vinorelbine, telithromycin, taxotere, basic fuchsin, and mixtures thereof.

Cytostatic agents are selected, for example, from: paclitaxel, lenalidomide, pomalidomide, epirubicin, 5-fluorouracil, sunitinib, lapatinib, canitinib, cyclophosphamide, hydroxyrubicin, lenalidomide/dexamethasone, pomalidomide/dexamethasone, carboplatin, mitoxantrone, oxaliplatin, docetaxel, vinorelbine, and any combination thereof.

The anti-angiogenic agent is selected from one or more of the following: bevacizumab, sorafenib, sunitinib, albesipine, IMC-1C11, betalain, N- (2, 3-dihydro-3, 3-dimethyl-1H-indol-6-yl) -2- [ (4-pyridylmethyl) amino ] -3-pyridinecarboxamide (AMG 706), 3- (4-bromo-2, 6-difluoro-benzyloxy) -5- [3- (4-pyrrolidin-1-yl-butyl) -ureido ] -isothiazole-4-carboxylic acid amide, pazopanib, N- (4- (3-amino-1H-indazol-4-yl) phenyl) -N' - (2-fluoro-5-methylphenyl) urea, or sildenib.

The vascular damaging agent is selected from combretastatin A4 and combretastatin A4 phosphate, and the inhibitor of nitric oxide synthase is selected from the group consisting of: NG-nitro-L-arginine, NG-methyl-L-arginine, NG-amino-L-arginine, L-N6- (1-iminoethyl) -ornithine, L-N6- (1-iminoethyl) -lysine, L-thiocitrulline, L-homochiocitrulline, S-methyl-L-thiocitrulline and 2-amino-4-methylpyridine.

In the treatment of tumors, such combination therapy may be achieved by simultaneous, sequential or separate administration of the various therapeutic ingredients. Such combination products employ the compounds of the invention in an effective dosage range and other pharmaceutically active agents in a permissible dosage range.

The invention has the following beneficial effects:

the inventor finds that the phenylalanine derivative is easy to prepare and good in drug-forming property, and can activate cancer-inhibiting p53 signal transduction at the cellular and animal level and inhibit tumor growth. Specifically, phenylalanine inhibits the ubiquitination-proteasome degradation process of wild-type p53 by regulating the interaction of wild-type p53 and HDM2, increases p53 protein stability, upregulates p53 signal transduction, and inhibits tumor growth.

The phenylalanine derivative has good application prospect in preparing antitumor drugs.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a scheme and a nuclear magnetic resonance spectrum of compound M-7;

FIG. 2 is a graph showing the results of antiproliferative activity of compound M-7 on tumor cells;

FIG. 3 is a graph showing the effect of compound M-7 on tumor cell clone formation;

FIG. 4 is a graph showing the results of a test for inhibiting tumor growth in mice by Compound M-7;

FIG. 5 is a graph showing the results of an inhibition test of Compound M-7 on primary liver cancer;

FIG. 6 is a graph of the results of assays for compounds M-7 inhibiting HDM2-p53 interactions;

FIG. 7 is a graph showing the results of an experiment of the effect of compound M-7 on the p53 signaling pathway;

FIG. 8 is a graph showing the results of antiproliferative activity of compound M-7-A on tumor cells;

FIG. 9 is a graph showing the results of antiproliferative activity of compound M-7-E on tumor cells.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); enzymatic methods (Methods in Enzymology) (Academic Press, inc.); experimental immunology handbook (Handbook of Experimental Immunology) (d.m. weir and c.c. blackwell); gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.Calos. Ed., 1987); current protocols for molecular biology (Current Protocols in Molecular Biology) (F.M. Ausubel et al, 1987); PCR, polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994); and contemporary immunological methods (Current Protocols in Immunology) (J.E. Coligan et al, 1991), each of which is expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The preparation of the compounds is carried out in this example:

the compound M-7 is prepared from diethyl dithiocarbamate and tyrosine derivative serving as raw materials through condensation reaction and hydrolysis reaction. Specifically, tyrosine derivative (ethyl 3- (4-acetylphenyl) -2-aminopropionate, 1 equivalent), triethylammonium diethyldithiocarbamate (1.2 equivalent), EDC (1.5 equivalent) and DMAP (0.2 equivalent) were taken and dissolved in a sufficient amount of anhydrous DMF to allow each component to be sufficiently dissolved, stirred at room temperature overnight, and quenched by adding saturated sodium bicarbonate solution. Extracting with chloroform for three times, mixing organic phases, concentrating by rotary evaporation to obtain crude product, and purifying by silica gel column chromatography. The structure of the product is confirmed by nuclear magnetic resonance, and the preparation route and the nuclear magnetic resonance spectrum are shown in figure 1.

Example 2

This example performs a cell proliferation assay of compound M-7 on tumor cells:

compound M-7 was tested for antiproliferative activity on tumor cells. Tumor cancer cells in logarithmic growth phase were inoculated into 96-well culture plates, cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, and then samples to be tested were added at different concentrations. After culturing at 37℃for 72 hours in a carbon dioxide incubator, the absorption value was measured by MTT method in an ELISA reader, and a cell viability curve was prepared. The test results are shown in FIG. 2.

FIG. 2 shows the antiproliferative effect of compound M-7 on tumor cells, and the result shows that compound M-7 has good antiproliferative activity on p53 wild-type tumor cells (A549, hepG2 and SK-Hep-1), but is insensitive to p53 function-obtaining mutation and function-losing mutation tumor cells.

Example 3

The effect of compound M-7 on the clonal formation of tumor cells was investigated in this example.

The effect of compound M-7 on tumor cell clone formation was tested. Tumor cells in the logarithmic growth phase were inoculated into 6-well plates (1000 cells/well), cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, then the samples to be tested were added, and after 7 days of culture in a carbon dioxide incubator at 37 ℃, stained with 0.5% crystal violet for 20 minutes, the staining solution was removed, and the image was recorded with a high resolution scanner, and the test results are shown in fig. 3. The results show that M-7 only works on tumor cells (SK-Hep-1, MCF-7 and HepG 2), but has no significant effect on normal cells (HEK-293T and B2B).

Example 4

This example describes the inhibition of tumor growth in mice by compound M-7.

The effect of compounds M-7, disulfiram (DSF) and Sorafenib (Sorafenib) on tumor growth in mice was tested by subcutaneously inoculating SK-Hep-1 hepatoma cells in nude mice. Tumor cells were collected in the logarithmic growth phase and washed with serum-free medium, and cells were resuspended in matrigel, after which 100. Mu.L of the cell suspension was injected subcutaneously into female BALB/c mice (5 weeks old). In tumor volume of 150-200mm ³ Thereafter, all mice were randomly divided into 4 groups and were treated with drug (2 mg/kg/3 days) and vehicle by intravenous injection, respectively, and tumor growth was recorded every 5 days (note: tumor volume was calculated as (ab) ² X 0.52 (a, long diameter; b, short diameter) and euthanized nude mice after 25 doses, tumors were dissected and recorded and the test results recorded as shown in fig. 4. Effect of compound on tumor growth in mice at a dose of 2 mg/kg. The result shows that the compound M-7 can obviously inhibit the growth of the tumor of mice. Photographs of tumor growth inhibition after dissection are shown in fig. 4.

Example 5

This example investigated the inhibition of primary liver cancer by compound M-7.

The Alb-cre/c-myc gene positive transgenic primary liver cancer mice with the age of 8 weeks are selected, the mice are administrated by tail vein injection, the administration dosage of M-7 is 2mg/kg, the survival condition of the primary liver cancer mice is observed, and a survival curve is drawn. The experimental result is shown in figure 5, and the M-7 drug can prolong the survival time of Alb-cre/c-myc transgenic mice and has remarkable anti-tumor capability.

Example 6

This example investigated the effect of compound M-7 on HDM2-p53 interactions.

Recombinant expression of HDM2 protein, and whether M-7 interacted with HDM2 was tested by isothermal titration experiments. The protein and compound buffers were HEPES (35 mmol/L) with 5% DMSO. HDM2 (20 μm) was titrated with M-7 (200 μm) in an isothermal titration calorimeter, and the titration curve was fitted using data analysis software to obtain the binding constant (K), binding enthalpy (Δh) and binding stoichiometry (n). All data were fitted using the single site binding model. The experimental results are shown in FIG. 6 (left), where M-7 can interact with HDM2 protein-small molecules. SK-Hep-1 cell lysates were incubated with M-7 and the effect of M-7 on HDM2-p53 interaction was tested by co-immunoprecipitation experiments. As shown in FIG. 6 (right), M-7 blocks the interaction of HDM2-p53 in cells and up-regulates the abundance of p53 protein.

Example 7

This example investigated the effect of compound M-7 on the p53 signaling pathway.

Tumor cells (HCT-116 and SK-Hep-1) and normal cells (HEK-293T) were treated with different concentrations of M-7, nutlin-3a (n 3 a) and vehicle and the abundance of p53, p21 and GAPDH in the cells was analyzed by Western Blotting. The results of the experiment are shown in FIG. 7, where M-7 was able to up-regulate the levels of p53 and p21 in tumor cells in a concentration-dependent manner, activating the p53 signaling pathway, but without significantly affecting the abundance of GAPDH.

Example 8

This example performs a cell proliferation assay of compound M-7-A on tumor cells:

compound M-7-a was tested for antiproliferative activity on tumor cells. Tumor cancer cells in logarithmic growth phase were inoculated into 96-well culture plates, cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, and then samples to be tested were added at different concentrations. After culturing at 37℃for 72 hours in a carbon dioxide incubator, the absorption value was measured by MTT method in an ELISA reader, and a cell viability curve was prepared. The test results are shown in FIG. 8, and the compound M-7-A has good antiproliferative activity on p53 wild-type SK-Hep-1 tumor cells.

Example 9

This example performs a cell proliferation assay of compound M-7-E on tumor cells:

compounds M-7-E were tested for antiproliferative activity on tumor cells. Tumor cancer cells in logarithmic growth phase were inoculated into 96-well culture plates, cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, and then samples to be tested were added at different concentrations. After culturing at 37℃for 72 hours in a carbon dioxide incubator, the absorption value was measured by MTT method in an ELISA reader, and a cell viability curve was prepared. The test results are shown in FIG. 9, and the compound M-7-E has good antiproliferative activity on p53 wild-type SK-Hep-1 tumor cells.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A phenylalanine derivative, which is a compound represented by any one of the following structural formulas:

、/>and->。

2. A pharmaceutical composition comprising the phenylalanine derivative according to claim 1.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable additive.

4. Use of a phenylalanine derivative according to claim 1 or a pharmaceutical composition according to any one of claims 2-3 for the preparation of an antitumor drug.

5. The use according to claim 4, wherein the anti-tumor drug is a p53 signaling drug.

6. The use according to claim 5, wherein the tumour is selected from malignant tumours.

7. The use according to claim 6, wherein the tumour is selected from liver cancer, breast cancer, cervical cancer, colon cancer, melanoma, lung cancer, ovarian cancer, gastric cancer, renal cancer or bladder cancer.

8. The use according to claim 6, wherein the tumour is selected from liver cancer, breast cancer, non-small cell lung cancer, cervical cancer or colon cancer.

9. Use of the phenylalanine derivative according to claim 1 or the pharmaceutical composition according to any one of claims 2-3 for the preparation of a medicament for the treatment of tumors, characterized in that said use is for:

10. A pharmaceutical composition, comprising: the phenylalanine derivative according to claim 1 or the pharmaceutical composition according to any one of claims 2-3, and a combination drug.

11. The pharmaceutical composition of claim 10, wherein the combination is at least one of the following: antiproliferative or antineoplastic agents for medical oncology, cytostatic agents, antiinvasive agents, inhibitors of growth factor function, antiangiogenic agents or vascular damaging agents.

12. The pharmaceutical composition of claim 11, wherein the antineoplastic agent is selected from the group consisting of: antimetabolites, alkylating agents, platinum salts, anthracyclines and intercalating agents, antimtopoisomerase, cytoskeletal agents, bleomycin, asparaginase, disulfiram and mixtures thereof.

13. The pharmaceutical composition of claim 11, wherein the antineoplastic agent is selected from the group consisting of methotrexate, 5-fluorouracil, fluorodeoxyuracil, cytarabine, 6-mercaptopurine, 6-thioguanine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, mitomycin C, aziridinyl benzoquinone, busulfan, carmustine, lomustine, fotemustine, carboplatin, daunorubicin, doxorubicin or doxorubicin, epirubicin, dactinomycin or actinomycin D, mitoxantrone, amsacrine, podophyllotoxin, etoposide, irinotecan, topotecan, vincristine, vinblastine, vindesine, vinorelbine, taxol, taxotere, basic fuchsin, and mixtures thereof.

14. The pharmaceutical composition of claim 11, wherein the cytostatic agent is selected from the group consisting of: paclitaxel, lenalidomide, pomalidomide, epirubicin, 5-fluorouracil, sunitinib, lapatinib, canitinib, cyclophosphamide, hydroxyrubicin, lenalidomide/dexamethasone, pomalidomide/dexamethasone, carboplatin, mitoxantrone, oxaliplatin, docetaxel, vinorelbine, and any combination thereof.

15. The pharmaceutical composition of claim 11, wherein the anti-angiogenic agent is selected from one or more of the following: bevacizumab, sorafenib, sunitinib, albesipine, IMC-1C11, betalain, N- (2, 3-dihydro-3, 3-dimethyl-1H-indol-6-yl) -2- [ (4-pyridylmethyl) amino ] -3-pyridinecarboxamide, 3- (4-bromo-2, 6-difluoro-benzyloxy) -5- [3- (4-pyrrolidin-1-yl-butyl) -ureido ] -isothiazole-4-carboxylic acid amide, pazopanib, N- (4- (3-amino-1H-indazol-4-yl) phenyl) -N' - (2-fluoro-5-methylphenyl) urea, or sildenib.

16. The pharmaceutical composition of claim 11, wherein the vascular damaging agent is selected from combretastatin A4 and combretastatin A4 phosphate.