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

Abstract

The invention discloses a phenylalanine derivative, a pharmaceutical composition and their application in tumor treatment, and relates to the technical field of anti-tumor pharmacy. A compound represented by the following structural formula (1) or a pharmaceutically acceptable salt thereof: Formula (1): Phenylalanine derivatives are easy to prepare and have good drug properties. They can activate tumor-suppressing p53 signal transduction at the cellular and animal levels and inhibit tumor growth. Specifically, phenylalanine derivatives regulate the interaction between wild-type p53 and HDM2, inhibit the ubiquitination-proteasome degradation process of p53, increase p53 protein stability, upregulate p53 signal transduction, and inhibit tumor growth. The above-mentioned phenylalanine derivatives have good preparation and anti-tumor drug application prospects.

Description

A kind of phenylalanine derivative, pharmaceutical composition and its application in tumor treatment

Technical field

The present invention relates to the technical field of anti-tumor pharmaceuticals, and specifically to a phenylalanine derivative, a pharmaceutical composition and their application in tumor treatment.

Background technique

The "World Health Statistics 2020" released by the World Health Organization (WHO) shows that approximately 9 million people worldwide die from malignant tumors every year, which 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 malignancy and poor prognosis, and have become a major public health issue that seriously threatens human health. Tumor evolution is a complex process involving multiple steps, pathways, and factors, involving dysfunction at multiple levels such as DNA, RNA, and proteins. At present, the treatment of malignant tumors remains a worldwide problem.

p53 is a key protein that regulates tumor cell apoptosis and cell cycle, and is an important tumor suppressor factor in the process of tumor evolution. Studies have shown that p53 is a target protein of the important oncogene HDM2, and the ubiquitination-proteasome degradation pathway mediated by HDM2 can regulate the stability and abundance of p53 in cells. The molecular regulatory network based on HDM2-p53 plays an important role in the malignant progression of tumors. It is necessary to find small molecules that can regulate p53 signal transduction to provide new strategies for the clinical treatment of tumors.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a phenylalanine derivative, a pharmaceutical composition and its application in tumor treatment to solve the above technical problems.

The present invention is implemented as follows:

In this specification, "tumor" refers to a population of cells that grows uncontrollably due to genetic mutations, including benign tumors and malignant tumors. In this specification, the term "malignant tumor" and the term "cancer" are used interchangeably. The term "cancer" broadly includes carcinomas that are epithelial origin cancers, sarcomas, and hematological malignancies such as leukemias. Examples of cancers derived from epithelial cancers include gastric cancer, colorectal cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, duodenal cancer, kidney cancer, prostate cancer, ovarian cancer, uterine cancer, breast cancer, and skin cancer. , hepatocellular carcinoma, tongue cancer, esophageal cancer, throat cancer, etc., but are not limited to these. Examples of sarcoma include fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, sarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, osteosarcoma, etc., but not Limited to this. Examples of hematological malignant tumors include, but are not limited to, leukemia, malignant lymphoma, multiple myeloma, and the like. In this specification, "tumor cells" refer to cells that form tumors, and typically refer to cells that proliferate abnormally regardless of surrounding normal tissues (so-called cancerized cells).

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

Formula 1): Wherein, R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, aryl group, acyl group, carboxylic acid group, carboxylic acid ester group, sulfonic acid group, sulfonic acid ester group, sulfinic acid group, sulfinic acid Ester group, nitrile group, alkyl group, alkenyl group, alkynyl group, halogen, alkoxy group, amino group, amine group, thioether group, nitro group, nitroso group, substituted alkyl group, substituted aryl group, penta One-membered heterocycle, six-membered heterocycle, silicon group, phosphonate group or phosphonate ester group.

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

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

The inventors found that the above-mentioned phenylalanine derivatives are easy to prepare, have good medicinal properties, and can activate tumor-suppressing p53 signal transduction at the cellular and animal levels and inhibit tumor growth. Specifically, phenylalanine derivatives regulate the interaction between wild-type p53 and HDM2, inhibit the ubiquitination-proteasome degradation process of wild-type p53, increase p53 protein stability, up-regulate p53 signal transduction, and inhibit tumor growth.

In other embodiments, the above-mentioned pharmaceutically acceptable compounds are pharmaceutically acceptable salts, and "pharmaceutically acceptable salts" include pharmaceutically acceptable inorganic acid salts or organic acid salts.

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

Optionally, the above-mentioned inorganic acid salts can be hydrohalic acid salts, halogen series oxygen-containing inorganic acid salts, carbon, nitrogen, and boron series oxygen-containing inorganic acid salts, phosphorus series inorganic acid salts, sulfur series inorganic acid salts, hydrocyanic acid Salt, chromate, dichromate, manganate, permanganate, molybdate or tungstate.

The hydrohalide salt may be hydrofluoride, hydrochloride, hydrobromide or hydroiodide, etc.

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

Carbon, nitrogen, and boron series oxygen-containing inorganic salts can be carbonates, nitrites, hyponitrites, nitrates, metanitrates, borates, (proto)borates, perborates, metabolites, etc. Silicates or (original) silicates, etc.

Phosphorus series inorganic acid salts can be metaphosphite, metaphosphate, (ortho)phosphate, phosphite, pyrophosphate, pyrophosphite, hypophosphite, per(one)phosphate, per(two) Phosphate or even (one) phosphate, etc.

Sulfur series inorganic acid salts can be hydrosulfate, sulfite, sulfate, per(mono)sulfate, thiosulfite, thiosulfate, dithionate, pyrosulfate or peroxydisulfate, etc. .

Optionally, the organic acid salt can be acetate, maleate, fumarate, succinate, citrate, p-toluenesulfonate, tartrate, formate, propionate, heptanoic acid Salt, oxalate, benzoate, malonate, succinate, maleate, hydroxybutyrate, citrate, mesylate, benzenesulfonate, lactic acid salt or mandelate.

Optionally, the above-mentioned organic acid salt may also be an organic acid salt compound containing functional groups such as carboxyl group, sulfonic acid group, sulfinic acid, sulfurcarboxylic acid, etc.

In other alternative ways, the above-mentioned inorganic acid salts and organic acid salts are not limited to the scope defined in the present invention.

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

In an alternative embodiment, the cycloalkyl group is selected from C _3-8 cycloalkyl. For example, selected from: C ₃ , C ₄ , C ₅ , C ₆ , C ₈ cycloalkyl.

In an optional embodiment, the above-mentioned non-cyclic alkyl group is selected from C ₁ -C ₈ alkyl group.

The above-mentioned alkenyl group is selected from -C ₁ -C ₈ alkenyl group and -C ₃ -C ₈ -cycloalkenyl group. The alkynyl group is selected from -C ₂ -C ₈ alkynyl.

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

In an optional embodiment, the phenylalanine derivative has any one of the following structural formulas:

The present invention also provides a pharmaceutical composition, which includes the above-mentioned phenylalanine derivative.

In an optional embodiment, the pharmaceutical composition further includes pharmaceutically acceptable additives or excipients.

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

In other embodiments, the pharmaceutical composition dosage form can also be other pharmaceutically acceptable dosage forms, and is not limited to the above protection scope.

In an alternative embodiment, sterile water or a water-propylene glycol solution can be used as a solvent to prepare a liquid formulation, and the active component can also be formulated in an aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active ingredient in water and adding suitable coloring agents, flavoring agents, stabilizers and thickening agents as necessary. Aqueous suspensions for oral use may be prepared by dispersing finely divided active ingredients in water together with viscous materials such as natural synthetic gums, resins, methyl cellulose, carboxymethyl cellulose and other agents known in the art. Suspending agent.

Pharmaceutical compositions may be in unit dosage form. In these forms, the composition is divided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation containing discrete quantities of the preparation, such as boxed tablets, capsules, and powders in vials. The unit dosage form can also be a capsule, cachet, or tablet, or it can be the appropriate number of any of these packaged forms.

The invention also provides the use of a phenylalanine derivative or pharmaceutical composition in the preparation of anti-tumor drugs.

In a preferred embodiment of the present invention, the above-mentioned anti-tumor drugs are drugs targeting p53 signal transduction.

In a preferred embodiment of the present invention, the above-mentioned tumors are 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, stomach cancer, kidney 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, the tumor is selected from the group consisting of lung cancer, breast cancer, colon cancer, midline cancer, stromal tumor, liver tumor, renal cancer, neuroma, adrenal carcinoma, acinar cell carcinoma, acoustic neuroma, acral carcinoma Lentigo melanoma, acrohidroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (acute myeloid leukemia), acute monocytic leukemia, acute progeroblastic leukemia Myeloid 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, alveolar soft tissue sarcoma, ameloblastoma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma , astrocytoma, atypical teratoma rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone Carcinoma, Brenner's tumor, Brown tumor, Burkitt's lymphoma, brain cancer, cancer cell, carcinoma in situ, carcinosarcoma, cartilaginous tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma , choroid plexus papilloma, renal clear cell sarcoma, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B Cell lymphoma, dysembryoplastic neuroepithelialoma, dysgerminoma, embryonal carcinoma, endocrine gland tumors, endodermal sinus tumors, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus within fetus, fibroma, fibrosarcoma , follicular lymphoma, follicular thyroid cancer, ganglioma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblast, giant cell tumor of bone, glial tumor, glioblastoma tumor, glioma, glioma, glucagonoma, gonadoblastoma, granulosa cell tumor, androcytoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer , hemangiopericytoma, malignant hematological diseases, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, invasive lobular carcinoma, intestinal cancer, renal cancer, laryngeal cancer, malignant Lentigo, fatal midline carcinoma, leukemia, Leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic 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 tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germline Cytoma, medullary breast carcinoma, medullary thyroid carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, myxoma , multiple myeloma, muscle tissue tumors, mycosis fungoides, myxoid sarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma , eye cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic neuroma, oral cancer, osteosarcoma, ovarian cancer, Pancost's tumor, papillary thyroid cancer, Paraganglioma, pineoblastoma, pineocytoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T lymphocyte lymphoma, primary central nervous system Systemic lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyomas, Rhabdomyosarcoma, Richter's transformation carcinoma, rectal cancer, sarcoma, Schwannomatosis tumor, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumor, Small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot warts, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestinal tumors, squamous cell carcinoma, gastric cancer, T Cellular lymphoma, testicular cancer, theca cell tumor, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, genitourinary cancer, urothelial cancer, uveal melanoma, uterine cancer, verrucous cancer, Visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

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

Inhibit the ubiquitination-proteasome degradation process of p53; increase p53 protein stability or upregulate p53 signal transduction.

The present invention also provides a pharmaceutical composition, which includes: a phenylalanine derivative or the above-mentioned pharmaceutical composition or combination drug.

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 anti-tumor compounds as the active ingredient.

In an optional embodiment, the above-mentioned combined drugs include, but are not limited to: chemotherapy drugs, radiotherapy, photosensitizers, photothermal agents, immunotherapy and other drugs.

The above-mentioned combined drug is at least one of the following drugs: anti-proliferation or anti-tumor drugs used in medical oncology, cell growth inhibitors, anti-invasive drugs, growth factor function inhibitors, anti-angiogenesis agents or blood vessel damage agents.

Antineoplastic drugs such as: folate antagonists, antimetabolites, alkylating agents, platinum salts, anthracyclines and insertion reagents, antitopoisomerases, drugs acting on the cytoskeleton, bleomycin, asparaginase , disulfiram and their mixtures.

The anti-tumor drugs are selected from methotrexate, 5-fluorouracil, fluorodeoxyuracil, cytarabine, 6-mercaptopurine, 6-thioguanine, methylchloroethylamine, cyclophosphamide, isophosphamide, Phalan, chlorambucil, thiotepa, mitomycin C, aziridinyl benzoquinone (AZQ), busulfan, carmustine (BCNU), lomustine (CCNU), fulan Mustine, carboplatin, daunorubicin, doxorubicin or doxorubicin, epirubicin, dactinomycin or actinomycin D, mitoxantrone, amsacridine, podophyllin, The group consisting of etoposide, irinotecan, topotecan, vincristine, vinblastine, vindesine, vinorelbine, Taxol, Taxotere, basic fuchsin and mixtures thereof.

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

The anti-angiogenic agent is selected from one or more of the following: bevacizumab, sorafenib, sunitinib, aflibercept, IMC-1C11, vatalanib, N-(2,3-di Hydrogen-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridylmethyl)amino]-3-pyridinecarboxamide (AMG706), 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 cediranib.

The vascular damaging agent is selected from comprestatin A4 and comprestatin A4 phosphate, and the inhibitor of nitric oxide synthase is selected from: 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-homothiocitrulline, S-methyl-L-thiocitrulline, and 2-amino-4-methylpyridine.

In treating tumors, this combination therapy can be accomplished by administering the various therapeutic components simultaneously, sequentially, or separately. Such combination products employ the compounds of the invention within an effective dosage range and the other pharmaceutically active agents within an approved dosage range.

The invention has the following beneficial effects:

The inventors found that the above-mentioned phenylalanine derivatives are easy to prepare, have good medicinal properties, and can activate tumor-suppressing p53 signal transduction at the cellular and animal levels and inhibit tumor growth. Specifically, phenylalanine regulates the interaction between wild-type p53 and HDM2, inhibits the ubiquitination-proteasome degradation process of wild-type p53, increases p53 protein stability, up-regulates p53 signal transduction, and inhibits tumor growth.

The above-mentioned phenylalanine derivatives have good application prospects in preparing anti-tumor drugs.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the preparation route and nuclear magnetic resonance spectrum of compound M-7;

Figure 2 is a graph showing the anti-proliferative activity results of compound M-7 on tumor cells;

Figure 3 is a diagram showing the effect of compound M-7 on tumor cell colony formation;

Figure 4 is a graph showing the results of compound M-7's tumor growth inhibition test in mice;

Figure 5 shows the results of the inhibition test of compound M-7 on primary liver cancer;

Figure 6 is a graph showing the test results of compound M-7 inhibiting HDM2-p53 interaction;

Figure 7 is a graph showing the test results of the effect of compound M-7 on the p53 signaling pathway;

Figure 8 is a graph showing the anti-proliferative activity results of compound M-7-A on tumor cells;

Figure 9 is a graph showing the results of the anti-proliferative activity of compound M-7-E on tumor cells.

Detailed ways

Reference will now 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. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in 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 in another embodiment, to yield still further embodiments.

Unless otherwise indicated, the practice of the invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait Editor, 1984); "Animal Cell Culture" (edited by R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Experimental Immunity" "Handbook of Experimental Immunology" (edited by D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (edited by J.M. Miller and M.P. Calos, 1987); "Current Molecular Biology" Methods (CurrentProtocols in Molecular Biology)" (F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction)" (Mullis et al., 1994); and "Contemporary Immunology 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 objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

The features and performance of the present invention will be described in further detail below with reference to examples.

Example 1

In this example, the preparation of the compound is carried out:

Compound M-7 was prepared through condensation reaction and hydrolysis reaction using diethyldithiocarbamate triethylammonium salt and tyrosine derivatives as raw materials. Specifically, take tyrosine derivative (ethyl 3-(4-acetoxyphenyl)-2-aminopropanoate, 1 equivalent), diethyldithiocarbamate triethylammonium salt (1.2 equivalent), EDC (1.5 equivalent) and DMAP (0.2 equivalent) was dissolved in sufficient anhydrous DMF to fully dissolve each component. Stir at room temperature overnight. Add saturated sodium bicarbonate solution to quench the reaction. Extract with chloroform three times, combine the organic phases, and concentrate by rotary evaporation to obtain a crude product, which is purified by silica gel column chromatography. The product structure was confirmed by NMR, and the preparation route and NMR spectrum are shown in Figure 1.

Example 2

This example conducts a cell proliferation test of compound M-7 on tumor cells:

Compound M-7 was tested for antiproliferative activity on tumor cells. Tumor cancer cells in the logarithmic growth phase are inoculated into a 96-well culture plate and cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, then add different concentrations of samples to be tested. After culturing in a carbon dioxide incubator at 37°C for 72 hours, use the MTT method to measure the absorption value in a microplate reader and prepare a cell viability curve. The test results are shown in Figure 2.

Figure 2 shows the anti-proliferative effect of compound M-7 on tumor cells. The results show that compound M-7 has good anti-proliferative activity on p53 wild-type tumor cells (A549, HepG2 and SK-Hep-1), while it has no effect on p53 function. Acquired mutations and loss-of-function mutations render tumor cells insensitive.

Example 3

This example studies the effect of compound M-7 on the clonogenic formation of tumor cells.

Compound M-7 was tested for its effect on tumor cell clonogenesis. Take tumor cells in the logarithmic growth phase, inoculate them into a 6-well culture plate (1000 cells/well), and culture them in DMEM (or RPMI1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours. Finally, add the sample to be tested, incubate it in a carbon dioxide incubator at 37°C for 7 days, stain it with 0.5% crystal violet for 20 minutes, remove the staining solution, and record the image with a high-resolution scanner. The test results are shown in Figure 3. The results showed that M-7 only had an effect on tumor cells (SK-Hep-1, MCF-7 and HepG2), but had no obvious effect on normal cells (HEK-293T and B2B).

Example 4

In this example, compound M-7 was tested for inhibiting tumor growth in mice.

Nude mice were subcutaneously inoculated with SK-Hep-1 liver cancer cells, and the effects of compound M-7, disulfiram (DSF) and sorafenib on tumor growth in mice were tested. Tumor cells were collected during the logarithmic growth phase, washed with serum-free medium, and resuspended in Matrigel, and then 100 μL of cell suspension was injected subcutaneously into female BALB/c mice (5 weeks old). After the tumor volume reached ^150-200mm3 , all mice were randomly divided into 4 groups and treated with intravenous injection of drug (2mg/kg/3 days) and vehicle respectively, and the tumor growth was recorded every 5 days (Note: Tumor The volume was calculated as (ab) ² × 0.52 (a, long diameter; b, short diameter), and the nude mice were euthanized 25 days after administration. The tumors were dissected, collected, and photographed to record the test results as shown in Figure 4. The compound was 2 mg /kg dose. The results show that compound M-7 can significantly inhibit tumor growth in mice. Photos of tumor growth inhibition after dissection are shown in Figure 4.

Example 5

This example studies the inhibitory effect of compound M-7 on primary liver cancer.

8-week-old Alb-cre/c-myc gene-positive transgenic primary liver cancer mice were selected and administered via tail vein injection. M-7 was administered at a dose of 2 mg/kg. The survival of the primary liver cancer mice was observed and Draw a survival curve. The experimental results are shown in Figure 5. The addition of M-7 can prolong the survival period of Alb-cre/c-myc transgenic mice and has significant anti-tumor ability.

Example 6

This example studies the effect of compound M-7 on HDM2-p53 interaction.

HDM2 protein was recombinantly expressed, and whether M-7 interacts with HDM2 was tested through isothermal titration experiments. Protein and compound buffers were HEPES (35 mmol/L) containing 5% DMSO. Titrate HDM2 (20 μM) with M-7 (200 μM) in an isothermal titration calorimeter and fit the titration curve using data analysis software to obtain the binding constant (K), binding enthalpy (ΔH) and binding stoichiometry (n). All data were fit using a single-site binding model. The experimental results are shown in Figure 6 (left). M-7 can interact with HDM2 in protein-small molecule interactions. SK-Hep-1 cell lysates were taken and incubated with M-7, and the effect of M-7 on HDM2-p53 interaction was tested through co-immunoprecipitation experiments. The experimental results are shown in Figure 6 (right). M-7 can block the intracellular HDM2-p53 interaction and upregulate p53 protein abundance.

Example 7

This example studies 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 (n3a) and vehicle, and p53, p21 and p53 in the cells were analyzed by Western Blotting. Abundance changes of GAPDH. The experimental results are shown in Figure 7. M-7 can upregulate the levels of p53 and p21 in tumor cells in a concentration-dependent manner and activate the p53 signaling pathway, but has no significant effect on the abundance of GAPDH.

Example 8

This example conducts a cell proliferation test of compound M-7-A on tumor cells:

Compound M-7-A was tested for antiproliferative activity against tumor cells. Tumor cancer cells in the logarithmic growth phase are inoculated into a 96-well culture plate and cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, then add different concentrations of samples to be tested. After culturing in a carbon dioxide incubator at 37°C for 72 hours, use the MTT method to measure the absorption value in a microplate reader and prepare a cell viability curve. The test results are shown in Figure 8. Compound M-7-A has good anti-proliferative activity against p53 wild-type SK-Hep-1 tumor cells.

Example 9

This example conducts a cell proliferation test of compound M-7-E on tumor cells:

Compound M-7-E was tested for antiproliferative activity against tumor cells. Tumor cancer cells in the logarithmic growth phase are inoculated into a 96-well culture plate and cultured in DMEM (or RPMI 1640) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 24 hours, then add different concentrations of samples to be tested. After culturing in a carbon dioxide incubator at 37°C for 72 hours, use the MTT method to measure the absorption value in a microplate reader and prepare a cell viability curve. The test results are shown in Figure 9. Compound M-7-E has good anti-proliferative activity against p53 wild-type SK-Hep-1 tumor cells.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (16)

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:

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

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.