WO2022188347A1 - 小分子化合物及其用途和组合物 - Google Patents

小分子化合物及其用途和组合物 Download PDF

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WO2022188347A1
WO2022188347A1 PCT/CN2021/110612 CN2021110612W WO2022188347A1 WO 2022188347 A1 WO2022188347 A1 WO 2022188347A1 CN 2021110612 W CN2021110612 W CN 2021110612W WO 2022188347 A1 WO2022188347 A1 WO 2022188347A1
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tert
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acetyl
glycyl
phenoxy
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胡延维
张熠
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Suzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/54—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the fields of biotechnology and medicine, and in particular relates to a small molecular compound that specifically inhibits NAC1 protein through design and synthesis based on bioinformatics and computer technology.
  • the present invention also relates to a pharmaceutical composition containing the small molecule compound, and discloses a method for using the compound for the treatment of diseases, especially the use in the treatment of malignant tumors.
  • the BTB/POZ family gene NACC1 gene encodes the expression of cellular transcription factor NAC1 (nucleus accumbens-1, nucleus accumbens 1), which is located in the human chromosome region Ch19p13.2 and is a newly discovered new carcinogen. NAC1 is generally highly expressed in various gynecological tumors (such as ovarian cancer, cervical cancer, endometrial cancer, breast cancer, etc.), but not in normal tissues.
  • the BTB domain (also known as the POZ domain) is an important domain that mediates protein interactions, and the BZB domain (1-129 amino acid sequence) of NAC1 is required for the formation of the NAC1 dimer complex, which forms the NAC1 dimer Aggregate complexes can participate in the regulation of various biological functions, such as anti-apoptosis, pro-proliferation, pro-invasion and metastasis, and anti-aging.
  • NAC1 is involved in inhibiting the apoptosis of ovarian cancer cells induced by cisplatin and paclitaxel, and the escape of apoptotic signals is closely related to the occurrence and development of tumors. Since NAC1 is only highly expressed in tumor cells and is involved in tumor resistance to apoptotic signals, small-molecule compounds targeting this molecule may be able to abolish its apoptosis-inhibiting function, and may be combined with cisplatin, paclitaxel, etc. Combination of chemotherapeutic drugs opens up a new strategy for tumor treatment.
  • the object of the present invention is to provide a compound that inhibits tumor cell growth or induces tumor cell apoptosis, and another object of the present invention is to provide a pharmaceutical composition prepared from the compound.
  • Another technical scheme of the present invention is:
  • NAI- 008 4-((2-(4-(tert-butyl)phenoxy)acetyl)glycyl)phenylbenzo[d][1,3]dioxane-5-carboxylate (NAI- 008), or its pharmaceutically, nutraceutical or food science acceptable salts or esters or derivatives, or their mixtures in the preparation of drugs for treating tumors, inhibiting tumor cell growth, and/or inducing tumor cell apoptosis use in.
  • the tumor or tumor cell is a tumor or tumor cell expressing human nucleus accumbens 1, and the expression level of human nucleus accumbens 1 of the tumor or tumor cell is 30%-50% higher than that of normal cells.
  • the tumor is selected from the following group: breast cancer, lung cancer, gastric cancer, prostate cancer, ovarian cancer, colon cancer, liver cancer, cervical cancer, endometrial cancer or B lymphoid tumors, and preferably the tumor is selected from the following group : ovarian cancer, cervical cancer, breast cancer, endometrial cancer; more preferably the tumor is selected from the group consisting of ovarian cancer.
  • Another technical scheme of the present invention is:
  • composition comprising:
  • apoptotic drugs such as carboplatin, doxorubicin, tamoxifen, 5-fluorouracil, difuran-fluorouracil, harringtonine, cytarabine, flutamide, ifosfamide, deoxyfluoride Uridine, loboplatin, letrozole, teniposide, angiostatin, endostatin or Avastin, etc., more preferably cisplatin or doxorubicin.
  • the weight ratio between the carboxylate and the apoptotic drug is 1:1000 to 1000:1, preferably the weight ratio is 1:500-500:1, more preferably the weight ratio is 1:100-100:1, more preferably the weight ratio 1:50-50:1.
  • Carboxylic acid esters comprise 1-95 wt %, preferably 5-90 wt %, more preferably 10-80 wt % of the total weight of the composition.
  • the dosage form of the composition is tablet, capsule, powder, granule, suspension or injection.
  • the content of the compound, its pharmaceutically or nutraceutically acceptable salt or ester, or their mixture is 0.05-50000mg/dose, preferably 0.1-10000mg / dose, more preferably 0.5-5000 mg / dose.
  • the small molecule compound designed for the human NAC1 protein of the present invention can effectively target and inhibit the anti-apoptotic molecule-NAC1 protein, so as to be used for cancer treatment;
  • the small molecule compound of the present invention can also be combined with other drugs and treatment methods for the treatment of malignant tumors;
  • the small molecule compound of the present invention has the advantages of good permeability, small toxic and side effects, simple structure and easy synthesis.
  • Figure 1 shows that the compound NAI-008 can inhibit the formation of NAC1 protein dimer
  • Figure 2A shows that the compound NAI-008 enhances the cell growth inhibition of cisplatin and doxorubicin, wherein the concentration of the compound used is 20 ⁇ M and the concentration of cisplatin and doxorubicin is 0-40 ⁇ M;
  • Figure 2B shows that NAI-008 enhances the cell proliferation inhibition of cisplatin and doxorubicin, where the concentration of the compound used is 20 ⁇ M and the concentration of cisplatin and doxorubicin is 0-40 ⁇ M;
  • Figure 2C shows that the compound NAI-008 enhanced cisplatin- and doxorubicin-induced apoptosis in a compound concentration of 20 ⁇ M and a cisplatin concentration of 20 ⁇ M.
  • Compound NAI-008 a small molecule compound 4-((2-(4-(tert-butyl)phenoxy)acetyl)glycyl)phenyl, which strongly enhances the inhibitory effect of cisplatin on the proliferation of ovarian cancer cell line SKOV3 Benzo[d][1,3]dioxane-5-carboxylate.
  • NAC1 is selectively highly expressed in ovarian cancer tumor tissue, but not expressed in normal ovarian epithelial tissue, suggesting that NAC1 may be closely related to tumorigenesis and growth.
  • overexpression of NAC1 can significantly resist cisplatin-induced apoptosis and promote cell growth, suggesting that it may be an anti-apoptotic molecule.
  • inhibiting the expression of NAC1 can enhance the sensitivity of ovarian cancer cells to cisplatin-induced apoptosis and inhibit the ability of tumor cells to form clones.
  • NAC1 is likely to be a target for the treatment of ovarian cancer.
  • the inventor's research shows that the anti-apoptotic NAC1 molecule plays an important role in the process of cell growth regulation, cell apoptosis, tumor occurrence and development, and the like. Therefore, NAC1 is likely to serve as a potential candidate target in the diagnosis and treatment of clinical tumors.
  • the small molecule compound specifically targets the anti-apoptotic molecule-NAC1 protein at the protein level , intervene in the biological behavior of tumor cells expressing NAC1, thereby effectively inhibiting the function of NAC1 and achieving anti-tumor effect.
  • small molecule compound of the present invention As used herein, the terms “small molecule compound of the present invention”, “compound NAI-008” or “compound of the present invention”, “m-amidobenzamide derivative of the present invention” are used interchangeably and all refer to small molecule compounds 4-((2-(4-(tert-butyl)phenoxy)acetyl)glycyl)phenylbenzo[d][1,3]dioxane-5-carboxylate and its pharmacy Acceptable salts and active derivatives of the above.
  • NAC1 4-((2-(4-(tert-butyl)phenoxy)acetyl)glycyl)phenylbenzo[d][1,3]dioxane-5-carboxylate is a
  • the lead compound targeting human nucleus accumbens 1 protein (NAC1) has the following structural formula:
  • the above compound is a brand new compound, which can be prepared by conventional organic synthesis methods.
  • a preferred "active ingredient” refers to a small molecule compound that can bind to human NAC1 protein, and that combined with cisplatin can reduce the growth of ovarian cancer SKOV3 cells to 50% % or less (ie, at least a 50% reduction), preferably to 38% or less, more preferably to 35% or less, or to a range between the above values as endpoints.
  • the m-amidobenzamide derivatives used in the present invention may be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases.
  • These salts include, but are not limited to: salts with inorganic acids such as hydrochloric, sulfuric, nitric, or phosphoric; salts with organic acids such as acetic, oxalic, succinic, or maleic; and others Salts, including but not limited to salts formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium.
  • a particularly preferred class of salts are the sodium or potassium salts.
  • the present invention also includes the compounds of the present invention in the form of esters (eg, carbamates) or other conventional "prodrugs" that, when administered in this form, are converted to the active moiety in vivo.
  • the small molecule compound of the present invention can effectively inhibit the function of human NAC1 protein, thereby inhibiting the proliferation of tumor cells and promoting the apoptosis of tumor cells.
  • the small molecule compound of the present invention targets human NAC1 protein at the molecular level, and reverses the biological behavior of tumor cells that positively express human NAC1 protein.
  • inhibiting the expression and function of NAC1 can inhibit the proliferation of tumor cells and promote tumor cell apoptosis
  • inhibiting the expression and function of NAC1 in vivo can inhibit the tumorigenicity of tumor cells in vitro
  • inhibiting The expression and function of NAC1 can inhibit the growth of tumor cells in vivo.
  • the present invention also includes pharmaceutical compositions containing meta-amidobenzamide derivatives and pharmaceutically acceptable salts or esters thereof.
  • the m-amidobenzamide derivatives and the pharmaceutical compositions thereof of the present invention can be used for treating cancer tumors, that is, administering a safe and effective amount of the m-amidobenzamide derivatives to mammals.
  • the compounds of the present invention can be used in combination with other chemotherapeutic agents, such as paclitaxel, carboplatin, doxorubicin, tamoxifen, 5-fluorouracil, difurofluorouracil, harringtonine, cytarabine, flutamide, ifosfamide , deoxyfluridine, loboplatin, letrozole or teniposide, etc.; tumor angiostatic drugs, such as angiostatin, endostatin, Avastin, etc., can control, alleviate or cure diseases by inhibiting NAC1 treatment, such as ovarian cancer and other cancers.
  • the compounds of the present invention can also be used in combination with anti-tumor traditional Chinese medicines (or their preparations).
  • a preferred pharmaceutical composition also contains an apoptotic drug, such as cisplatin and the like.
  • the m-amidobenzamide derivative or a pharmaceutically acceptable salt or ester thereof When used to treat tumors, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents etc. to form a pharmaceutical composition.
  • pharmaceutically acceptable carriers or excipients such as solvents, diluents etc.
  • Liquid carriers include sterile water, polyethylene glycols, nonionic surfactants, and edible oils (eg, corn oil, peanut oil, and sesame oil).
  • Solid carriers include starch, lactose, dibasic calcium phosphate, microcrystalline cellulose, sucrose and kaolin, as appropriate to the identity of the active ingredient and the particular mode of administration desired.
  • Adjuvants commonly used in the preparation of pharmaceutical compositions may also advantageously be included, such as flavors, colors, preservatives and antioxidants such as vitamin E, vitamin C, 2,6-di-tert-butyl-p-cresol (BHT) and tert-butylhydroxyanisole (BHA).
  • compositions of the present invention include the following dosage forms: Oral administration dosage forms: such as tablets, capsules, dispersible powders, granules or suspensions (suspensions) (containing, for example, about 0.05-5% suspending agents (cosolvents) ), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol); or as sterile injectable solutions or suspensions (containing about 0.05-5% in an isotonic medium) cosolvent) for parenteral administration.
  • Oral administration dosage forms such as tablets, capsules, dispersible powders, granules or suspensions (suspensions) (containing, for example, about 0.05-5% suspending agents (cosolvents) ), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol); or as sterile injectable solutions or suspensions (containing about 0.05-5% in an isotonic medium) cosolvent) for parenteral administration
  • These pharmaceutical preparations may generally contain about 0.001-99.9 wt%, preferably 0.5-99.5 wt%, preferably 2.5-90 wt%, more preferably 5-60 wt% by weight of the active ingredient (m-amide group) mixed with a carrier.
  • benzamide derivatives or pharmaceutically acceptable salts or esters thereof based on the total weight of the composition.
  • these compounds of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although pH can vary depending on the nature of the substance being formulated and the condition being treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral or topical administration. Intravenous administration is preferred.
  • m-amidobenzamide derivatives used in the present invention can also be administered parenterally or intraperitoneally.
  • Solutions or suspensions of these active compounds can also be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquids, polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, these forms must be sterile and must be fluid for easy syringe expelling. It must be stable under the conditions of manufacture and storage and must be resistant to the contaminating influence of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, alcohol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • N-acetylated phenoxyacetamide derivatives of the present invention can also be used in combination with other tumor treatment methods (such as radiotherapy) or other therapeutic agents (such as cisplatin, doxorubicin, etc.).
  • tumor treatment methods such as radiotherapy
  • other therapeutic agents such as cisplatin, doxorubicin, etc.
  • the effective dose of active ingredient employed may vary with the mode of administration and the severity of the disease to be treated. Typically, however, when a compound of the present invention is administered at a daily dose of about 0.01-100 mg/kg animal body weight (preferably 0.02-20 mg/kg body weight, more preferably 0.1-10 mg/kg body weight), the For satisfactory results, it is preferably administered in a dose of 1-4 times a day, or in a sustained release form. For most large mammals, the total daily dose is about 5-5000 mg or more, preferably 10-1000 mg. Dosage forms suitable for oral administration contain about 0.5-500 mg of the active compound in admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen can be adjusted to provide the best therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
  • the preferred pharmaceutical compositions are liquid compositions. Intravenous administration of the meta-amidobenzamide derivatives is preferred.
  • m-amidobenzamide derivatives or pharmaceutically acceptable salts or esters or extracts thereof can also be used for preparing health care compositions for adjuvant therapy of tumors.
  • the health care composition contains a safe and effective amount (such as 0.01-99 wt%) of m-amidobenzamide derivatives or their acceptable salts or esters of health care products, or extracts and health care products. acceptable carrier.
  • the health care product composition of the present invention may contain the same content of the m-amidobenzamide derivative or its salt, ester or extract that is acceptable for health care products as the pharmaceutical composition.
  • the content of the mid-amidobenzamide derivative in the health care product composition can be slightly lower, for example, containing 0.01-50 wt% of the mid-amidobenzamide derivative or its pharmaceutically acceptable salt or ester.
  • the health care product composition of the present invention can be made into any conventional formulations by conventional methods, preferably tablet formulations, oral liquid formulations, granules and capsule formulations.
  • m-amidobenzamide derivatives or their food acceptable salts or Esters or extracts are used to prepare food additives, so as to be added to food, improve the anti-tumor ability of the subject and assist in the treatment of tumors.
  • the food additive may contain a safe and effective amount (such as 0.01-99 wt %) of the m-amidobenzamide derivative or a food-acceptable salt or ester or extract thereof, and a food- acceptable Carrier.
  • the food additive of the present invention may contain the same content of the m-amidobenzamide derivative or its food acceptable salt, ester or extract as the pharmaceutical composition or the health care product composition.
  • the content of the intermediate amidobenzamide derivative of the food additive can be lower than that in the health care product, for example, containing 0.01-50 wt% of the intermediate amidobenzamide derivative or a food acceptable salt or ester thereof.
  • the food additive of the present invention can be made into any conventional form by conventional methods, such as solution, powder, syrup and the like.
  • SKOV3 cell line purchased from ATCC, an ovarian cancer tumor cell line that positively expresses NAC1.
  • the culture method of SKOV3 cell line is as follows: the cells were inoculated in DMEM (InVitrogen) medium containing 10% calf serum, and placed in a 37°C, 5% CO2 incubator for routine culture. The medicine was cultured for two weeks.
  • DMEM InVitrogen
  • the dimer structure of the POZ domain of NAC1 was obtained from the RCSB PDB, the binding mode of the dimer was analyzed, and the surface shape and hydrophilic and hydrophobic properties of the receptor were calculated using the SiteFinder function of MOE to determine the most suitable small molecule inhibitor. More than 10 designed compounds were pretreated for the combined active sites, and the ADMET properties of the compounds were filtered using Oprea's leadlikeness filter to retain molecules suitable as lead compounds. Energy minimization was performed for each compound separately, and then rigid and flexible docking methods were used in turn to dock small molecules to the aforementioned active sites of the receptors, respectively. To calculate the binding free energy, take molecules with binding energies below -10 kcal/mol. Calculate the molecular topological fingerprints, perform cluster analysis according to the Tanimoto similarity between fingerprints, comprehensively score and structure diversity to extract structural diversity subsets, and select the final candidate based on the visual judgment of the receptor-ligand interaction mode molecular.
  • NAI-008 small molecule compound (4-((2-(4-(tert-butyl)phenoxy)acetyl)glycyl)phenylbenzo[d][1,3]dioxane
  • the structural formula of -5-carboxylate is shown in formula I:
  • NAI-008 The synthetic route of the small molecule compound (NAI-008) is as follows:
  • p-tert-butylphenol as raw material, it is condensed with ethoxyacetyl chloride under alkaline conditions, and hydrolyzed under alkaline conditions to free the carboxyl group.
  • the carboxyl group is condensed with p-methoxyphenylacetamide under the catalysis of EDCI, and further under the action of BBr3 Removal of methyl protection, the free phenolic hydroxyl and carboxylic acid to form esters to obtain the target molecule.
  • Small molecule compound (NAI-008) can inhibit the dimer formation of target NAC1 protein.
  • the dimer formation of the target NAC1 protein by small molecule compounds was detected by immunoprecipitation technology and immunoblotting.
  • This embodiment adopts the conventional MTT method.
  • the specific method is as follows:
  • SKOV3 cells were plated overnight in 96-well plates at a density of 3000 cells/well.
  • the small molecule compound NAI-008 was then added to the wells at various concentrations (1.25, 2.5, 5, 10, 20, 50 ⁇ M) alone or in combination with a final concentration of 20 ⁇ M cisplatin.
  • 10 ⁇ l of MTT 5 mg/ml was added to each well, and the supernatant was discarded after incubation at 37 degrees Celsius for 4 hours.
  • After dissolving the purple crystal with 150 ⁇ l of dimethyl sulfoxide it was placed in a microplate reader (Bio-Rad Company) to detect the absorbance at 570 nm.
  • SKOV3 cells were treated for 24-48 hours according to the experimental requirements, digested with trypsin, and the cell suspension was repeatedly pipetted to fully disperse the cells. The percentage of single cells should be above 95% and counted on a hemocytometer. According to the concentration of 2000 cells per dish, 2 ml of cell suspension was inoculated into the culture dish to make the cells evenly dispersed. Culture in 37°C, 5% CO 2 for 7 to 14 days, with fresh medium from time to time. Observe under an inverted microscope, when macroscopic colonies appear in the petri dish, terminate the culture, discard the medium, and rinse twice with PBS. Crystal violet staining for 20 minutes. The results were analyzed after 2-3 washes with PBS.
  • NAI-008 could significantly enhance the inhibition of cell growth and proliferation by cisplatin and doxorubicin at a final concentration of 20uM, while its single application did not cause significant toxic effects on cells, as shown in Figures 2A and 2B.
  • the ovarian cancer cell line SKOV3 cells were plated into 24-well plates at a density of 20,000 cells/well, and after 16-24 hours of incubation, NAI-008 was added to a final concentration of 20 ⁇ M. After 4 hours, additional cisplatin or doxorubicin was added to a final concentration of 20 ⁇ M. After 48 hours, the expression of PARP cleaved by apoptotic protein was detected by immunoblotting.
  • Figure 2C the expression of apoptotic proteins was significantly higher in cells treated with cisplatin or doxorubicin 20 ⁇ M and NAI-008 20 ⁇ M than in cells treated with cisplatin or doxorubicin alone. Furthermore, consistent with the MTT results: the compound alone did not cause significant apoptosis.
  • the compound of the present invention can specifically inhibit the formation of human nucleus accumbens 1 protein dimer, so it can be used to treat tumors with high expression of this protein, such as ovarian cancer.
  • results of the above examples of the present invention show that the compounds of the present invention can stably inhibit NAC1, and synergize with cisplatin or doxorubicin to kill tumor cells.
  • 2-(4-tert-butylphenoxy)-N-(2- ⁇ [(4-tert-butylphenoxy)acetyl]amino ⁇ ethyl)acetamide and NAI-008 It is two compounds with completely different core structures.
  • the former uses acylated ethylenediamine as the main effect structure, and the latter uses acylated phenoxyacetamide as the main effect structure.
  • the relationship between the two is not a simple derivative. A completely different type of structure.
  • the present invention discloses an anti-tumor small molecule compound targeting human nucleus accumbens 1 and a composition thereof, which can effectively target and inhibit the anti-apoptotic molecule-NAC1 protein, so as to be used for cancer treatment It can also be combined with other drugs and treatment methods for the treatment of malignant tumors; it has the advantages of good permeability, small toxic and side effects, simple structure and easy synthesis.

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Abstract

本发明公开了小分子化合物及其用途和组合物，所述小分子化合物的用途具体为：4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物用于制备治疗肿瘤、抑制肿瘤细胞生长、和/或诱导肿瘤细胞凋亡的物质，所述的组合物含有：(1)化合物4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物；(2)致凋亡药物；(3)药学、保健品学、或食品学上可接受的载体或赋形剂。

Description

小分子化合物及其用途和组合物 技术领域
本发明属于生物技术和医学领域，具体涉及一种基于生物信息学和计算机技术，经设计、合成得到的特异性抑制NAC1蛋白的小分子化合物。本发明还涉及含有该小分子化合物的药物组合物，并公开了将该化合物药物用于疾病治疗的方法，特别是在恶性肿瘤的治疗中的用途。
背景技术
恶性肿瘤是危害人类健康的一类疾病。随着肿瘤分子生物学、基因组学、蛋白组学的发展，大量在肿瘤发生发展中起关键作用的调控因子被揭示。在此基础上，抗肿瘤药物研发理念的重大转变。研发焦点正从传统细胞毒药物向针对肿瘤发生发展过程中关键调控因子方向发展，这些靶点新药针对正常细胞和肿瘤细胞之间的差异，可达到高选择性、低毒性的治疗效果，从而克服传统细胞毒药物的选择性差、毒副作用强、易产生耐药性等缺点，为此，肿瘤靶向治疗学进入了一个崭新的研发阶段。近年来，随着分子生物学、X射线晶体学的发展，大量与肿瘤相关的生物大分子的三维结构被确定；计算科学的快速发展，计算机辅助药物设计应运而生，并渗透到新药研发的各个环节，极大地提高药物研发的成功率，降低研发成本，缩短研发周期，目前已成为创新药物研究的核心技术之一。
BTB/POZ家族基因NACC1基因编码细胞转录因子NAC1(nucleus accumbens-1，伏隔核1)的表达，其定位于人染色体区Ch19p13.2，是新近发现的一个新的致癌因子。NAC1在多种妇科肿瘤中(例如卵巢癌、宫颈癌、子宫内膜癌、乳腺癌等)普遍高表达，而在正常组织未见表达。BTB结构域(也称为POZ结构域)是介导蛋白相互作用的重要结构域，NAC1的BZB结构域(1-129氨基酸序列)是形成NAC1二聚体复合物所必需的，形成的NAC1二聚体复合物可以参与多种生物功能调控：例如抗凋亡、促增殖、促侵袭转移、抗衰老等。
有实验表明，NAC1参与抑制顺铂、紫杉醇引起的卵巢癌细胞凋亡，而凋亡信号的逃逸与肿瘤的发生发展密切相关。既然NAC1只是特异性地在肿 瘤细胞高表达，而且参与了肿瘤对凋亡信号的抵抗，因此针对该分子的小分子化合物可能能够取消其凋亡抑制功能，并将有可能和顺铂、紫杉醇等化疗药物联用，开辟一条肿瘤治疗新策略。
目前，虽然已经开发了一些抑制肿瘤生长的化合物，然而人们仍需要开发新的具有抑制肿瘤生长或诱导肿瘤细胞凋亡的化合物。
发明内容
本发明目的是：提供一种具有抑制肿瘤细胞生长或诱导肿瘤细胞凋亡的化合物，本发明的另一目的是提供由所述化合物制成的药物组合物。
本发明的技术方案是：
4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯，其结构式如下：
Figure PCTCN2021110612-appb-000001
其合成路线为：
Figure PCTCN2021110612-appb-000002
本发明的另一技术方案是：
4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯(NAI-008)、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物在制备用于治疗肿瘤、抑制肿瘤细胞生长、和/或诱导 肿瘤细胞凋亡的药物中的用途。
进一步的，所述的用途在于制备人伏膈核1的抑制剂。
进一步的，所述的肿瘤或肿瘤细胞是表达人伏膈核1的肿瘤或肿瘤细胞，所述肿瘤或肿瘤细胞的人伏膈核1表达量高于正常细胞30％～50％。
进一步的，所述肿瘤选自下组：乳腺癌、肺癌、胃癌、前列腺癌、卵巢癌、结肠癌、肝癌、宫颈癌、子宫内膜癌或B淋巴系肿瘤，较佳的肿瘤选自下组：卵巢癌、宫颈癌、乳腺癌、子宫内膜癌；更佳的肿瘤选自下组：卵巢癌。
本发明的另一技术方案是：
提供—种组合物，所述的组合物含有：
(1)化合物4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物；
(2)致凋亡药物或肿瘤血管抑制药物；
(3)药学、保健品学、或食品学上可接受的载体或赋形剂。
进一步的，所述的致凋亡药物如卡铂、阿霉素、三苯氧胺、5-氟尿嘧啶、双呋喃氟尿嘧啶、三尖杉酯碱、阿糖胞苷、氟他胺、异环磷酰胺、脱氧氟尿苷、洛波钼、来屈唑、替尼泊甙、血管他丁、内皮他丁或阿瓦斯丁等，更优选顺铂或阿霉素。
进一步的，所述的4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯和致凋亡药物之间重量比为1:1000至1000:1，优选重量比为1:500-500:1，更优选重量比为1:100-100:1，再优选重量比为1:50-50:1。
进一步的，所述的4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯占组合物总重量的l-95wt％，优选5-90wt％，更优选10-80wt％。
进一步的，所述组合物的剂型是片剂、胶囊、粉末剂、颗粒剂、混悬剂或注射剂。在所述组合物为单位剂型或多剂型时，其中所述化合物、其药学上或保健品学上可接受的盐或酯、或它们的混合物的含量为0.05-50000mg/剂，优选0.1-10000mg/剂，更优选0.5-5000mg/剂。
本发明的优点是：
(a)本发明针对人NAC1蛋白所设计的小分子化合物，可有效靶向抑制抗凋亡分子----NAC1蛋白，从而用于癌症治疗；
(b)本发明的小分子化合物还可以与其它药物和治疗手段联合，用于恶性肿瘤的治疗；
(c)本发明的小分子化合物具有渗透性好，毒副作用小，结构简单，易于合成等优点。
附图说明
图1显示了化合物NAI-008可抑制NAC1蛋白二聚体的形成；
图2A显示了化合物NAI-008增强顺铂和阿霉素的细胞生长抑制，其中所用化合物浓度为20μM，顺铂和阿霉素浓度为0-40μM；
图2B显示了NAI-008增强顺铂和阿霉素的细胞增殖抑制，其中所用化合物浓度为20μM，顺铂和阿霉素浓度为0-40μM；
图2C显示了化合物NAI-008增强顺铂和阿霉素诱导的细胞凋亡，其中所用化合物浓度为20μM，顺铂浓度为20μM。
具体实施方式
经过广泛而深入的研究，发现部分肿瘤细胞内NAC1的表达往往升高，抑制NAC1的表达和功能可以抑制肿瘤细胞的增殖和体内外的致瘤活性。为此，本发明人借助计算机辅助药物设计并合成出了若干个可能会和NAC1结合的小分子化合物，随后运用体外结合实验和MTT的方法对这些化合物进行了第二轮筛选，首次得到一个可以强烈增强顺铂对卵巢癌细胞系SKOV3增殖抑制作用的化合物NAI-008，即小分子化合物4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯。
具体而言，本发明人的研究表明，NAC1在卵巢癌肿瘤组织中的选择性高表达，而在正常卵巢上皮组织中未见表达，提示NAC1可能与肿瘤发生及生长密切相关。在低表达NAC1的ES2卵巢癌细胞系内，NAC1的过表达可明显抵抗顺铂诱导的凋亡，促进细胞生长，提示其可能是一个具有抗凋亡作用的分子。在高表达NAC1的卵巢癌肿瘤细胞内，抑制NAC1的表达可增强 卵巢癌细胞对顺铂诱导凋亡的敏感性，抑制肿瘤细胞的克隆形成能力。提示NAC1很可能是治疗卵巢癌的作用靶点。本发明人的研究表明，抗凋亡作用的NAC1分子在细胞生长调控、细胞凋亡、肿瘤发生发展等过程中发挥重要的作用。因此，NAC1很可能在临床肿瘤的诊断和治疗中作为潜在的候选靶标。
在此基础上，本发明人设计合成了大量的化合物，从而获得了一种可有效抑制肿瘤的小分子化合物，所述的小分子化合物特异性地在蛋白水平靶向抗凋亡分子—NAC1蛋白，对表达NAC1的肿瘤细胞的生物学行为进行干预，从而有效抑制NAC1的功能，达到抗肿瘤效果。
下面具体介绍：
如本文所用，术语“本发明的小分子化合物”、“化合物NAI-008”或“本发明化合物”、“本发明的间酰胺基苯甲酰胺衍生物”可互换使用，都指出小分子化合物4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯及其药学上可接受的盐和活性衍生物。
4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯是一种靶向于人伏隔核1蛋白(NAC1)的先导化合物，其结构式如下：
Figure PCTCN2021110612-appb-000003
上述化合物是一种全新化合物，可用常规的有机合成方法制备获得。
1、活性成分
在本发明中，优选的“活性成分”指这样的小分子化合物：所述的化合物与人NAC1蛋白能够相互结合，并且所述的化合物与顺铂联合可使卵巢癌SKOV3细胞的生长降低到50％以下(即降低至少50％)，优选降低到38％以下，更优选降低到35％以下，或降低到以以上数值为端点的范围之间。
本发明中所用的间酰胺基苯甲酰胺衍生物可以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)：与如下无机酸形 成的盐：如盐酸、硫酸、硝酸、或磷酸；与如下有机酸形成的盐，如乙酸、草酸、丁二酸或马来酸；以及其它盐，包括但不限于：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐。一类特别优选的盐是钠盐或钾盐。
本发明还包括以酯(例如氨基甲酸酯)或其它常规的“前体药物”的形式(当以这种形式给药时，在体内可转化成活性部分)存在的本发明化合物。
本发明的小分子化合物能有效抑制人NAC1蛋白的功能，从而抑制肿瘤细胞的增殖，促进肿瘤细胞的凋亡。具体地，本发明的小分子化合物在分子水平针对人NAC1蛋白，对阳性表达人NAC1蛋白的肿瘤细胞的生物学行为进行逆转。实验已证明：(1)抑制NAC1的表达和功能可抑制肿瘤细胞的增殖，促进肿瘤细胞凋亡；(2)体内抑制NAC1的表达和功能可抑制肿瘤细胞的体外致瘤性；(3)抑制NAC1的表达和功能可抑制肿瘤细胞体内的生长。
2、药物组合物
本发明还包括含有间酰胺基苯甲酰胺衍生物及其药学上可接受的盐或酯的药物组合物。本发明的间酰胺基苯甲酰胺衍生物及其药物组合物可用于治疗癌症肿瘤，即给哺乳动物施用安全有效量的间酰胺基苯甲酰胺衍生物。
本发明化合物可与其它化疗药联用，如紫杉醇、卡铂、阿霉素、三苯氧胺、5-氟尿嘧啶、双呋喃氟尿嘧啶、三尖杉酯碱、阿糖胞苷、氟他胺、异环磷酰胺、脱氧氟尿苷、洛波钼、来屈唑或替尼泊甙等；肿瘤血管抑制药物，如血管他丁、内皮他丁、阿瓦斯丁等，可通过抑制NAC1来控制、缓解或治愈疾病的治疗，例如卵巢癌等癌症。此外，本发明的化合物还可与抗肿瘤的中药(或其制剂)合用。
一种优选的药物组合物还含有致凋亡药物，如顺铂等。
当间酰胺基苯甲酰胺衍生物或其药学上可接受的盐或酯用于治疗肿瘤时，它可与一种或多种药学上可接受的载体或赋形剂混合，如溶剂、稀释剂等，从而形成药物组合物。
液态载体包括：无菌水、聚乙二醇、非离子型表面活性剂和食用油(如玉米油、花生油和芝麻油)。固态载体包括：淀粉、乳糖、磷酸氢钙、微晶纤维素、蔗糖和白陶土，只要适合活性成分的特性和所需的特定给药方式。在制备药物组合物中通常使用的佐剂也可有利地被包括，例如调味剂、色素、 防腐剂和抗氧化剂如维生素E、维生素C、2,6-二叔丁基对甲酚(BHT)和叔丁基羟基茴香醚(BHA)。
通常，本发明的药物组合物包括以下剂型：口服给药剂型：如片剂、胶囊、可分散的粉末、颗粒或悬浮液(混悬剂)(含有如约0.05-5％悬浮剂(助溶剂))、糖浆(含有如约10-50％糖)、和酏剂(含有约20-50％乙醇)；或者以无菌可注射溶液或混悬剂形式(在等渗介质中含有约0.05-5％助溶剂)进行非肠胃道给药。这些药物制剂通常可含有与载体混合的约0.001-99.9wt％，优选0.5-99.5wt％，较佳地2.5-90wt％，更佳地5％-60wt％(重量)的活性成分(间酰胺基苯甲酰胺衍生物或其药学上可接受的盐或酯)，按组合物的总重量计。
在制备药物组合物时，通常，可将这些本发明的化合物配制于无毒的、惰性的和药学上可接受的水性载体介质中，其pH通常约为5-8，较佳的pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、肌内、腹膜内、静脉内、皮下、皮内、口服或局部给药。优选静脉给药方式。
本发明所用的间酰胺基苯甲酰胺衍生物也可肠胃外或腹腔内给药。也可在适当混合有表面活性剂(如羟丙基纤维素)的水中制备这些活性化合物(作为游离碱或药学上可接受的盐)的溶液或悬浮液。还可在甘油、液体、聚乙二醇及其在油中的混合物中制备分散液。在常规储存和使用条件下，这些制剂中含有防腐剂以防止微生物生长。
适应于注射的药物形式包括：无菌水溶液或分散液和无菌粉(用于临时制备无菌注射溶液或分散液)。在所有情况中，这些形式必须是无菌的且必须是流体以易于注射器排出流体。在制造和储存条件下必须是稳定的，且必须能防止微生物(如细菌和真菌)的污染影响。载体可以是溶剂或分散介质，其中含有如水、醇(如甘油、丙二醇和液态聚乙二醇)、它们的适当混合物和植物油。
在使用本发明所述的N-乙酰化苯氧乙酰胺衍生物时，还可与其它肿瘤治疗手段(如放疗)或其它治疗剂(如顺铂、阿霉素等)联用。
所用活性成分的有效剂量可随给药的模式和待治疗的疾病的严重程度 而变化。然而，通常当本发明化合物每天以约0.01-100mg/kg动物体重(较佳地0.02-20mg/kg体重，更佳地0.l-l0mg/kg体重给药)的剂量给予时，能得到令人满意的效果，较佳地每天以1-4次的剂量给予，或以缓释形式给药。对大部分大型哺乳动物而言，每天的总剂量约为5-5000mg或更高，较佳地10-1000mg。适用于内服的剂量形式，包含与固态或液态药学上可接受的载体混合的约0.5-500mg的活性化合物。可调节此剂量方案以提供最佳治疗反应。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
从易于制备和给药的立场看，优选的药物组合物是液态组合物。间酰胺基苯甲酰胺衍生物的静脉给药是优选的。
3、保健品组合物
除了制备药物组合物用于治疗肿瘤之外，在本发明中，还可将间酰胺基苯甲酰胺衍生物或其保健品学上可接受的盐或酯或提取物用于制备保健品组合物，从而用于辅助治疗肿瘤。
在本发明中，保健品组合物含有安全有效量(如0.01-99wt％)的间酰胺基苯甲酰胺衍生物或其保健品学上可接受的盐或酯、或提取物和保健品学上可接受的载体。
本发明的保健品组合物可与药物组合物一样含有相同含量的间酰胺基苯甲酰胺衍生物或其保健品学上可接受的盐或酯或提取物。通常，保健品组合物中间酰胺基苯甲酰胺衍生物的含量可略低一些，例如含0.01-50wt％间酰胺基苯甲酰胺衍生物或其保健品学上可接受的盐或酯。
本发明的保健品组合物，可以通过常规方法制成任何常规的制剂形式，优选的是片剂、口服液、颗粒剂和胶囊制剂。
4、食品添加剂
除了制备药物组合物用于治疗肿瘤之外和作为保健品组合物用于辅助治疗肿瘤以外，在本发明中，还可将间酰胺基苯甲酰胺衍生物或其食品学上可接受的盐或酯或提取物用于制备食品添加剂，从而用于加入食品中，提高对象的抗肿瘤能力并辅助治疗肿瘤。
在本发明中，食品添加剂可含有安全有效量(如0.01-99wt％)的间酰胺基苯甲酰胺衍生物或其食品学学上可接受的盐或酯或提取物，和食品学上可 接受的载体。
本发明的食品添加剂可与药物组合物或保健品组合物一样含有相同含量的间酰胺基苯甲酰胺衍生物或其食品学上可接受的盐或酯或提取物。通常，食品添加剂中间酰胺基苯甲酰胺衍生物的含量可低于保健品中的含量，例如含0.01-50wt％间酰胺基苯甲酰胺衍生物或其食品学上可接受的盐或酯。
此外，在适当的情况下，将本发明的间酰胺基苯甲酰胺衍生物或其食品学上可接受的盐或酯或提取物直接作为食品添加剂使用也是可行的，只要它们不会影响食品的口味和/或外观。
本发明的食品添加剂，可以通过常规方法制成任何常规的形式，例如溶液、粉末、糖浆等。
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例，还应包括在本发明所要求的权利范围内其他任何公知的改变。
此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，《分子克隆：实验室手册》(纽约，冷泉港实验室出版社，New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
细胞系
SKOV3细胞系：购自ATCC，这是一种阳性表达NAC1的卵巢癌肿瘤细胞系。
SKOV3细胞系的培养方法如下：将细胞接种于含10％小牛血清的DMEM(InVitrogen公司)培养液中，置于37摄氏度、体积分数为5％的CO 2 培养箱中常规培养，实验前无药培养两周。
实施例
1、小分子化合物(NAI-008)的筛选
采用计算机辅助的虚拟筛选方法进行。
首先从RCSB PDB中获取NAC1的POZ domain的二聚体结构，分析二聚体的结合模式，使用MOE的SiteFinder功能对受体的表面形状和亲疏水性质等进行计算，确定最适合小分子抑制剂结合的活性位点，对设计的10多个化合物进行前处理，使用Oprea's leadlikeness filter对化合物的ADMET性质进行过滤，保留适合作为先导化合物的分子。对每个化合物分别进行能量最小化，然后依次采用刚性和柔性对接方式，将小分子分别对接到受体的前述活性位点。计算结合自由能，取结合能低于-10kcal/mol的分子。计算分子的拓扑结构指纹，根据指纹间的Tanimoto相似度进行聚类分析，综合打分与结构多样性提取结构多样性子集，结合对受体-配体作用模式的可视化判断，挑选出最终的候选分子。
其中，NAI-008小分子化合物(4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯)的结构式如式I所示：
Figure PCTCN2021110612-appb-000004
2、小分子化合物(NAI-008)的合成路线如下：
Figure PCTCN2021110612-appb-000005
以对叔丁基苯酚为原料，碱性条件下与乙氧基乙酰氯缩合，碱性条件下水解游离出羧基，羧基在EDCI催化下与对甲氧基苯乙酰胺缩合，进一步在BBr3作用下脱除甲基保护，游离出的酚羟基与羧酸成酯得目标分子。
3、小分子化合物(NAI-008)可抑制靶标NAC1蛋白的二聚体形成。
对于选出的NAI-008(自行合成)，通过免疫沉淀技术、免疫印迹法检测小分子化合物对靶标NAC1蛋白的二聚体形成。
结果证实NAI-008可抑制NAC1蛋白二聚体的形成，如图1所示。
4、小分子化合物(NAI-008)对肿瘤细胞的生长和增殖抑制
本实施例采用了常规的MTT法。具体方法如下：
将SKOV3细胞以3000个细胞/孔的密度铺96孔板培养过夜。然后，把小分子化合物NAI-008以不同浓度(1.25、2.5、5、10、20、50μM)单独或与20μM终浓度的顺铂联合加入孔中。48小时后，在每孔中加入MTT(5mg/ml)10μl，37摄氏度孵育4小时后吸弃上清。用150μl二甲基亚砜溶解紫色结晶后，置于酶标仪(Bio-Rad公司)，检测570nm处的吸光度。
本实施例采用了常规的克隆集落形成实验法。具体方法如下：
根据实验要求对SKOV3细胞进行处理24～48h后，用胰酶消化下来，细胞悬液反复吹打，使细胞充分分散，单个细胞百分率应在95％以上，在血球计数板上计数。按照每皿含2000个细胞的浓度分别接种2ml细胞悬液到培养皿中，使细胞分散均匀。37℃、5％CO 2中培养7～14天，中途不时更换新鲜培养基。在倒置显微镜下观察，待培养皿中出现肉眼可见的集落时，终止 培养，弃去培养基，使用PBS润洗两遍。结晶紫染色20分钟。PBS洗脱2-3次后分析结果。
结果显示，NAI-008可以在20uM终浓度显著增强顺铂和阿霉素对细胞生长和增殖的抑制，而其单独应用未对细胞造成显著的毒性作用，如图2A和2B所示。
5、小分子化合物(NAI-008)对顺铂和阿霉素杀伤肿瘤细胞的增强效应的检测
将卵巢癌细胞系SKOV3细胞以20000个/孔的密度铺到24孔板中，孵育16-24小时后，加入NAI-008至终浓度20μM。4小时后，再加入顺铂或阿霉素至终浓度20μM。作用48小时后，用免疫印迹法检测凋亡蛋白剪切的PARP表达。结果如图2C所示：联用了顺铂或阿霉素20μM和NAI-008 20μM的凋亡蛋白表达显著性高于单独用顺铂或阿霉素的细胞。此外，与MTT结果一致：单独应用化合物并未引起显著的细胞的凋亡。
本发明的化合物可特异性抑制人伏隔核1蛋白二聚体的形成，因此可用于治疗高表达该蛋白的肿瘤，如卵巢癌。
本发明上述的实施例的结果表明，本发明化合物可稳定地抑制NAC1，协同顺铂或阿霉素杀伤肿瘤细胞。
需说明的的是，2-(4-叔丁基苯氧基)-N-(2-{[(4-叔丁基苯氧基)乙酰基]氨基}乙基)乙酰胺与NAI-008是两个完全不同母核结构的化合物，前者以酰化的乙二胺为主要效应结构，后者是酰化的苯氧乙酰胺为主要效应结构，两者不是简单的衍生物的关系，属于完全不同的结构类型。
综上所述，本发明公开了一种靶向于人伏隔核1抗肿瘤小分子化合物及其组合物，可有效靶向抑制抗凋亡分子----NAC1蛋白，从而用于癌症治疗；还可以与其它药物和治疗手段联合，用于恶性肿瘤的治疗；具有渗透性好，毒副作用小，结构简单，易于合成等优点。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. 4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯，其结构式如下：
    Figure PCTCN2021110612-appb-100001
    其合成路线为：
    Figure PCTCN2021110612-appb-100002
  2. 4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物在制备用于治疗肿瘤、抑制肿瘤细胞生长、和/或诱导肿瘤细胞凋亡的药物中的用途。
  3. 根据权利要求2所述的用途，其特征在于：用于制备人伏膈核1的抑制剂。
  4. 根据权利要求2所述的用途，其特征在于：所述肿瘤细胞是表达人伏膈核1的肿瘤细胞，所述肿瘤细胞的人伏膈核1表达量高于正常细胞30％～50％。
  5. 根据权利要求2所述的用途，其特征在于：所述肿瘤细胞选自卵巢癌、宫颈癌、乳腺癌或子宫内膜癌。
  6. —种组合物，其特征在于，所述的组合物含有：
    (1)化合物4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯、或其药学、保健品学或食品学上可接受的盐或酯或衍生 物、或它们的混合物；
    (2)致凋亡药物；
    (3)药学、保健品学、或食品学上可接受的载体或赋形剂。
  7. 根据权利要求6所述的组合物，其特征在于，所述致凋亡药物为顺铂或阿霉素。
  8. 根据权利要求6所述的组合物，其特征在于，所述的4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯和致凋亡药物之间的重量比为1:50-50:1。
  9. 根据权利要求6所述的组合物，其特征在于，所述的4-((2-(4-(叔丁基)苯氧基)乙酰基)甘氨酰)苯基苯并[d][1,3]二氧杂环-5-羧酸酯占组合物总重量的10-80wt％。
  10. 根据权利要求6所述的组合物，其特征在于，所述组合物的剂型是片剂、胶囊、粉末剂、颗粒剂、混悬剂或注射剂。
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